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Research ArticleInterfering Expression of Chimeric TranscriptSEPT7P2-PSPH Promotes Cell Proliferation inPatients with Nasopharyngeal Carcinoma
JingWang12 Guo-Feng Xie12 Yuan He 12 Ling Deng2 Ya-Kang Long2
Xin-Hua Yang2 Jiang-JunMa2 Rui Gong12 Wen-Jian Cen2 Zu-Lu Ye2 Yi-Xin Zeng1
Hai-YunWang 12 and Jian-Yong Shao 12
1State Key Laboratory of Oncology in South China Collaborative Innovation Center for Cancer MedicineGuangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis andTherapy Sun Yat-sen University Cancer CenterNo 651 Dongfeng East Road Guangzhou 510060 China2Department of Molecular Diagnostics Sun Yat-sen University Cancer Center Guangzhou 510060 China
Correspondence should be addressed to Hai-Yun Wang wanghaiysysuccorgcn and Jian-Yong Shao shaojysysuccorgcn
Received 29 July 2018 Revised 9 January 2019 Accepted 3 February 2019 Published 1 April 2019
Academic Editor Akira Hara
Copyright copy 2019 Jing Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Introduction Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is mostly prevalent in southernChinaThe development of NPC involves accumulation of multiple genetic changes Chromosomal translocation is always thoughtto be accompanied with the fusion chimeric products To data the role of the fusion chimeric transcript remains obscureMaterials and Methods We performed RNA sequencing to detect the fusion genes in ten NPC tissues Sanger sequencingand quantitative RT-PCR were used to measure the level of the fusion chimeric transcript in NPC tissues and cell lines Thefunctional experiments such as CCK8 assay colony formation and migrationinvasion were conducted to analyze the role of thistranscript in NPC in vitro Results We demonstrated that the chimeric transcript SEPT7P2-PSPH was formed by trans-splicing ofadjacent genes in the absence of chromosomal rearrangement and observed in both NPC patients and cell lines in parallel Low-expression of the SEPT7P2-PSPH chimeric transcript induced the protein expression of PSPH and promoted cell proliferationmetastasisinvasion and transforming ability in vitro Conclusions Our findings indicate that the chimeric transcript SEPT7P2-PSPH is a product of trans-splicing of two adjacent genes and might be a tumor suppressor gene potentially having the role ofanticancer activity
1 Introduction
Nasopharyngeal carcinoma (NPC) presents regional differ-ences in incidence such as high incidences in North Africaand Southern China [1] More than 60 of NPC patients atdiagnosis are in late stages or even present with metastasisalthough many biomarkers were reported to be useful forearly detection such as Epstein-Barr virus (EBV) antibodies[2] Therefore there is still an urgent need to identify thebiomarkers for cancer diagnosis and therapeutic guidance
Gene fusions derived generally from chromosomal rear-rangements are considered as cancer-related genetic events
and their products including RNAs and proteins are iden-tified as specific diagnostic and therapeutic targets forcancers such as hematologic malignancy and lung adeno-carcinoma [3 4] However certain chimeric transcripts canbe formed without chromosomal rearrangements Previ-ous studies demonstrated that a fusion transcript namedSLC45A3-ELK4 was formed with exon 1 of SLC45A3 andthe last 4 exons of ELK4 [5 6] which acted as onco-genes in prostate tumor cells without chromosomal changes[7] Additionally many studies showed a plenty of fusiontranscripts present in normal andor tumor cells [8ndash12]Therefore fusion genes might be occurred in normal or
HindawiJournal of OncologyVolume 2019 Article ID 1654724 10 pageshttpsdoiorg10115520191654724
2 Journal of Oncology
tumors cells withwithout chromosomal changes Althoughprevious studies have already reported some tumor specificfusion genes in NPC all of them occur accompanied with therearrangement of chromosomes [13 14]
Herein we carried out RNA sequencing and discovered anovel type of fusion gene trans-splicing chimeric transcriptSEPT7P2-PSPH In addition we performed in vitro studiesto demonstrate that the ability of cell invasion migrationproliferation colony formation and transforming were acti-vated when the expression of the transcript was decreasedindicating that SEPT7P2-PSPH act as the tumor suppressorgene role in NPC tumorigenesis
2 Materials and Methods
21 Primary Tumors and Pair-End RNA Sequencing Tenformalin fixed paraffin embedded (FFPE) NPC samples wereretrieved from the Department of Pathology of Sun Yat-senUniversity Cancer Center (SYSUCCGuangzhou China) Alltumor samples were reviewed by two experienced patholo-gists Total RNA were extracted from ten FFPE tissues withRNeasy FFPE Kit (QIANGEN Hilden Germany) accordingto the manufacturerrsquos instructions for RNA sequencingcDNA libraries were prepared according to the standardprotocol provided by themRNA-seq sample Pre Kit (IlluminaInc California USA) and sequenced (90 nt paired-end) onthe Illumina Hi-seq 2000 To identify fusion genes frompaired-end RNA sequencing the data were analyzed bythe computational pipeline called SOAPfusion which usesclusters of discordant paired-end alignments to inform asplit-read alignment analysis for finding fusion boundaries[15] The UCSC H sapiens reference genome (build hg19)was used for alignments The written informed consent wasobtained from each patient and this study was approvedby Ethics Committee and Institutional Review Board ofSYSUCC
22 Cell Cultures Reverse Transcription and Sanger Sequenc-ing CNE1 and 6-10B used in the study were cultivated inRPMI 1640 (Invitrogen Carlsbad CA USA) supplementedwith 10 FBS (Gibco California USA) in a humidified 5CO2incubator at 37∘C All cultivations involved have been
sustained within 6 months Total RNA was extracted usingTrizol (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos instructions Reverse transcription was sub-sequently performed with PrimeScript RT reagent Kit withgDNA Eraser (TaKaRa Kusatsu Shlga Japan) The productswere employedwith specific primers for Sanger sequencing toconfirm potential fusion transcripts The primerswere shownas follows
SEPT7P2-PSPH 1-sense 51015840-TTACGACTTCTCGGT-CTTCGG-31015840
SEPT7P2-PSPH 1-anti-sense 51015840-CGTCCTCAACGC-CACAGATT-31015840
23 Quantitative RT-PCR Quantitative RT-PCR was per-formed using the ABI 7500 Real-time PCR system (Applied
Biosystems California USA) and SYBR Premix EX Taq(TaKaRa Kusatsu Shlga Japan) All reactions were carriedout in triplicate The housekeeping gene GAPDH was used asnormalization control The relative expression levels of targetgene SEPT7P2-PSPH were calculated by 2minus119862119905 method inwhich Ct = mean Ct SEPT7P2-PSPH ndash mean Ct controlwhere Ct values are the cycle threshold for each sample [16]Specific primers were shown as follows
SEPT7P2-PSPH 2-sense 51015840-ACCTGAGCCTGGGAG-GAAA-31015840
SEPT7P2-PSPH 2-anti-sense 51015840-GTCAACATCAAA-ACACACAGCATC-31015840
24 Immunohistochemistry (IHC) Staining 4120583m thick FFPENPC slides were used to perform IHC staining Firstly thetumor slideswere deparaffinized Antigen retrieval procedurewas then performed in a pressure cooker in 1mM sodium cit-rate (Sangon Biotech Shanghai China) for 15minutes PSPHrabbit polyclonal (Proteintech Wuhan China) was dilutedwith SigalStain antibody diluent (Cell Signaling TechnologyDanvers MA USA) at 1400 for 1 h Universal secondaryantibody (Gene Tech Shanghai China) was applied for 15minutes Subsequently diaminobenzidine was used as chro-mogens and slides were counterstained with haematoxylinbefore mounting PSPH IHC was scored according to thescoring criteria including intensity and percentage as follows0 no staining 1+ faint cytoplasmic reactivity without anybackground staining 2+ moderate cytoplasmic reactivityand 3+ granular cytoplasmic reactivity of strong intensity 00-25 1 25-50 2 50-75 [17]
25 siRNA Transfection Small interfering RNAs which tar-geted the fusion gene (si-SP1 51015840-GUUGUUTTTTCCTCC-CUGG-31015840 si-SP2 51015840-UUGUUTTTTCCTCCCUGGC-31015840)were bought from RIBOBIO Company (Guangzhou China)CNE1 and 6-10B cells were transfected with oligonucleotides(20 nmolL) using the LipofectamineRNAiMAX transfectionreagent (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos instructions
26WesternBlotting Cell lysiswas carried out in RIPAbufferincluding protease inhibitor and phosphatase inhibitor cock-tails (Biostool Shanghai China) The protein extracts wereisolated from the same amounts using 12 SDS-PAGE gelsAfter that all the abstracts were transferred to polyvinylidenefluoride membranes (Merck Millipore Billerica MA USA)Subsequently the membranes were incubated with rabbitpolyclonal anti-PSPH antibody 11000 (Proteintech WuhanChina) And then the incubation was performed with goatanti-rabbit IgG antibody 15000 (Abcam Cambridge UK)Anti-120573-actin antibody (Affinity Ancaster Canada) was actedas a protein control
27 Cell Proliferation Assay Cell proliferation was evaluatedby conducting CCK-8 assays 1000 cells were seeded per wellin 96-well plates in triplicate After incubation with 10uLof CCK8 agent (Dojindo Kumamoto Japan) for 3 hours in
Journal of Oncology 3
the dark in an incubator at 37∘C the absorbance value wasmeasured at 450 nm every 12 hours
28 Boyden Chamber Assays Invasion and migration abili-ties were examined after the siRNA of SEPT7P2-PSPH trans-fection Boyden chambers (Corning New York USA) with 8120583m inserts equipped with (invasion) or without (migration)Matrigel were placed in the 24-well plate A total of 5 times 104cells were suspended in 200 120583L RPMI 1640 without serumin the upper chambers and cultured at 37∘C for 24 hoursThe cells which went through the inserts were stained with05 crystal (Sangon Shanghai China) Then the numberof cells per field of view was counted under phase-contrastmicroscopy
29 Colony Formation Assay 500 transfected cells wereplated in six-well plates and cultured for 7 or 12 daysAll the colonies were fixed with 4 paraformaldehyde andstained with 05 crystal violet (Sangon Shanghai China)for quantification All the experiments were performed intriplicate
210 Anchorage-Independent Assay In the 6-well plate thebottom layers were the mixture of equivalent volumes of12 agarose (Sangon Shanghai China) in PBS and mediumcontaining 20 FBS 5000 cells were suspended in the upperlayer containing 1ml 04 soft agarose
211 Statistical Analysis All statistical analyses were per-formed using SPSS 160 software (IBM Armonk NY USA)and GraphPad Prism version 50 (GraphPad Software) Thedata are presented as the mean plusmn SEM from at leastthree independent experiments Two-tailed Studentrsquos t-testswere conducted to compare the difference between groupsProgression-free survival (PFS) and overall survival (OS)were calculated using the Kaplan-Meier method and the sur-vival curves were compared with the log-rank test methodAll tests were two-sided and P lt 005 was considered asstatistically significant
3 Results
31 Novel Fusion Genes Discovered in NPC We found 60transcripts of fusion genes using RNA sequencing including28 inter- and 32 intrachromosomal fusions from ten FFPENPC tissues (Table 1) Paired-end reads featured by twouncorrelated genes or spanned exon-exon junctions werenominated as putative chimeric genes (Figures 1(a) and 1(b))Fusion genes recurrently occurred in more than one samplewere selected for subsequent investigation A novel fusiongene named SEPT7P2-PSPH was discovered in 5 out of10 samples and located on chromosome 7 (Figure 1(b)) Tovalidate this newly found transcript we designed specificprimers matching the region that contained the fusion pointat the mRNA level The mRNA level of this fusion gene wasvalidated using Sanger sequencing in the 5 samples previouslyand consistent with that identified by RNA sequencing(Figure 1(c))
The transcript was formed with the exon 1 of SEPT7P2and the exon 4 of PSPH The practical distance betweenthe two exons are 102 Mb on chromosome 7 To estimateif there existed a genomic DNA rearrangement with thechimeric RNA we designed 30 primer pairs to amplify theentire genome sequences between exon 1 and exon 2 ofSEPT7P2 as well as the genome sequences between exon 3and exon 4 of PSPH (Supplementary Table 1 and Figure 1(d))However all the segments were amplified by PCR indirectlyindicating the fact that chromosomal arrangementwas absent(Figure 1(e))
32 The Expression of the Fusion Genes in Cell Lines andTissues In addition we have investigated several tumor celllines including Hela Hep3B NP69 CNE1 6-10B SUNE2CNE2 HONE1 and 5-8F All the cell lines were confirmed toharbor the fusion gene (Figure 2(a)) Interestingly we foundthat the relative expression of SEPT7P2-PSPH was higherin the cohort of nasopharyngitis than that in NPC patients(Figure 2(b) P lt 005) Collectively these results indicatedthat the newly found transcript was not peculiar to tumorcells Analogous studies have been published to demonstratethe noncanonical chimeric transcript [10ndash12] Simultane-ously we performed western blotting assay to discover ifthere would be a new type of fusion protein accompanied bythe transcript Anti-PSPH polyclonal antibody was appliedin the nine tumor cell lines It was shown that the putativefusion protein did not exist and only the protein of full-lengthPSPH was confirmed by western blot with the band at 25 kDa(Figure 2(c))
33 Knocking Down SEPT7P2ndashPSPH Upregulated the Levelof PSPH Protein and Promoted Cell Proliferation and Migra-tion We carried out in vitro experiments to confirm thepotential biologic function of the SEPT7P2-PSPH transcriptNPC cell lines CNE1 and 6-10B were selected to explorethe effect of SEPT7P2-PSPH on NPC tumorigenesis Whencompared with the negative control group the expressionof the chimeric transcript substantially decreased after twospecifically designed siRNAs siSP1 and siSP2 were tran-siently transfected into the cell lines Also there was nonovel fusion protein synthesized After transfection for 48hours the expression of SEPT7P2-PSPH at mRNA level wasdecreased sharply whereas the expression of PSPH proteinwas upregulated in both cell lines (Figures 2(d)-2(e) andSupplementary Figure 1) This may indirectly show thephenomenon that the chimeric RNA fusion did not producea new fusion protein
However the expression level of down-stream genersquosprotein was reversely influenced by the occurrence of thefusion between SEPT7P2 and PSPH We subsequently per-formed cell proliferation assays to explore the effects ofthe fusion gene on the growth of CNE-1 and 6-10B Thecells transfected with si-SEPT7P2ndashPSPH-1 (siSP1) and si-SEPT7P2ndashPSPH-2 (siSP1) grew faster than those with neg-ative control (Figure 3(a) P lt 005) Low expression ofSEPT7P2-PSPH may contribute to tumor cell proliferationFor investigating its influence on the transforming ability of
4 Journal of Oncology
(a)
(b)
SEPT7P2 PSPH
(c)
SEPT7P2
PSPH
Exon 1 Exon 2
Exon 3 Exon 4
(d)
SEPT7P2
PSPH
(e)
Figure 1 RNA sequencing was used to discover SEPT7P2-PSPH fusion gene in NPC (a) Detection of SEPT7P2-PSPH fusion gene via RNAsequencing The reads are aligned across the junction of the predicted fusion transcripts (b) Circos plot of SEPT7P2-PSPH fusion generevealed the fusion existing in chromosome 7 (c)The fusion breakpoint was verified using Sanger sequencing (d) A schematic illustration ofthe amplified regions of fusion gene with exons 1-2 of SEPT7P2 and exons 3-4 of PSPH (e)The sequences between exons 1 and 2 of SEPT7P2and the sequences between exons 3 and 4 of PSPH were all amplified by RTndashPCR
Journal of Oncology 5
Table 1 60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues
up gene up chr down gene down chr Fusion TypeIGKC chr2 CALR chr19 INTERCHR-DSIGKJ5 chr2 CALR chr19 INTERCHR-DSMALAT1 chr11 RFWD2 chr1 INTERCHR-DSMRPS18A chr6 C5orf25 chr5 INTERCHR-DSRN7SL1 chr14 IGKC chr2 INTERCHR-DSRN7SL1 chr14 IGKJ5 chr2 INTERCHR-DSRNU6-1 chr15 SNORD3A chr17 INTERCHR-DSRNU6-33 chr4 SNORD3A chr17 INTERCHR-DSRNU6-42 chr3 RPS3 chr11 INTERCHR-DSRNU6-42 chr3 SNORD15A chr11 INTERCHR-DSRNU6-42 chr3 SNORD3A chr17 INTERCHR-DSSAMD12 chr8 ATP10D chr4 INTERCHR-DSATP1B2 chr17 HSD17B12 chr11 INTERCHR-SSE2F4 chr16 RPL14 chr3 INTERCHR-SSEFCAB4A chr11 RN7SK chr6 INTERCHR-SSIGHG1 chr14 NINJ1 chr9 INTERCHR-SSIGKC chr2 CSNK2A2 chr16 INTERCHR-SSIGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SSMIDN chr19 ALMS1 chr2 INTERCHR-SSPDE4B chr1 PPP6R3 chr11 INTERCHR-SSPOU2AF1 chr11 PARP11 chr12 INTERCHR-SSRNU6-2 chr10 SNORD3A chr17 INTERCHR-SSRNU6-36 chr12 SNORD3A chr17 INTERCHR-SSRNU6-42 chr3 SNHG12 chr1 INTERCHR-SSRPL14 chr3 GLS chr2 INTERCHR-SSSLC7A5P2 chr16 PHC3 chr3 INTERCHR-SSSMARCA2 chr9 RPL14 chr3 INTERCHR-SSZNF827 chr4 ZNF318 chr6 INTERCHR-SSADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAPAP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAPC12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAPCOL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAPCTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAPCTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAPEEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAPJAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAPKIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAPLSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAPMAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAPMRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAPNPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAPPIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAPPOLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAPPPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAPPROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAPPSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAPRRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAPSTYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAPTTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAPVMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP
6 Journal of Oncology
Table 1 Continued
up gene up chr down gene down chr Fusion TypeWDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAPWNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAPEEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAPHACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAPIRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAPTMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAPRMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAPFGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGOSEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGOSRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGOchr chromosome
NPC cells we conducted both colony formation assay andanchorage-independent growth assay Strikingly CNE1 and6-10B cells with siSP1 and siSP2 formed larger colonies thanthe negative control groups which was reversely correlatedwith the expression of the chimeric RNA (Figures 3(b) and3(c) P lt 005) To evaluate its biological function withmigration and invasion Boyden chamber assays in vitrohave been conducted As shown in Figure 3(d) knock-ing down SEPT7P2-PSPH with siSP1 and siSP2 markedlyenhanced the migration and invasion of NPC cells (P lt005)
34 PSPH Expression and Prognosis of NPC Patients Toinvestigate the expression status of PSPH in NPC weperformed IHC experiment for PSPH in 72 FFPE NPCspecimens (Figure 4) The result revealed that the patientswith high PSPH expression showed a tendency with shorterPFS and OS in NPC patients though there was no statisticallysignificant (Figures 5(a) and 5(b) P = 0205 P = 0106)
4 Discussion
The present study is the first to identify trans-splicingchimeric transcript SEPT7P2-PSPH without chromosomalrearrangement in NPC patients Knocking down of this tran-scriptmay promote cell proliferation andmetastasisinvasionpossibly due to the upregulation expression of the down-stream gene PSPH
Traditionally chromosome rearrangements can generatefusion genes which are in an intimate relationship withtumor carcinogenesis To date it makes possible to discovermore and more valuable fusion genes acting as a crucialregulator in cancer formation maintenance and evolutionPrevious studies have shown that chimeric RNAs in normaland cancer cells can be generated by intergenic splicing inthe absence of chromosome rearrangements [7ndash9 18 19]This kind of fusion gene can be used as ldquononcanonicalchimerasrdquo It has been reported that there are two meth-ods for ldquononcanonical chimerasrdquo formation involving cis-splicing and trans-splicing of adjacent genes [10] Cis-splicing
fusions tend to occur between two genes that are locatedwithin 30kb in the same chromosome Trans-splicing canbe classified into two types intragenic trans-splicing takingplace between two pre-mRNAs transcribed from the samegenome loci and intergenic trans-splicing that is from twodifferent genome loci SEPT7P2-PSPH can be classified asintergenic trans-splicing located on chromosome 7 alignedwith the fact that the up- and downstream genes are 102Mbdistance
Furthermore we found that metastasisinvasion andproliferation of the tumor cells were inversely enhanced afterthe transcript had been knocked down This was differentfrom previously analogous studies [14 20] The westernblot result showed that there was no newly synthetic fusionprotein accompanied with the chimeric RNA Only the PSPHprotein was translated from the downstream gene PSPHand was overexpressed after the chimeric RNA had beenknocked downThe sequence of the chimeric RNA indicatedthat both of the two genesrsquo start codons were retained afterthe occurrence of the fusion one in exon 1 of SEPT7P2and another in exon 4 of PSPH It seemed that the startcodon in exon 1 of SEPT7P2 depressed the transcriptionof PSPH The reason why fusion genes have the ability ofoncogenesis can be illuminated that the new synthesizedfusion products possess the tumorigenic function Anotherreason is that the preoncogene could be upregulated afterconnected to a stronger promoter or the antioncogenecould be downregulated after fused with a weaker promoter[21]
SEPT7P2 is a pseudogene which has been proved tobe related to SEPT7 and expressed in all tissue types [22]playing a role in multiple biological processes includingvesicle trafficking apoptosis remodeling of the cytoskeletonneurodegeneration and neoplasia
Some researchers have reported that abnormal expressionof PSPH is strongly relevant to the hepatocellular carcinomapatientsrsquo mortality as well as the critical importance oncMyc-induced cancer progression both in vitro and in vivo[23] The level of PSPH has the relationship with breastcancer and lacrimal gland adenoid cystic carcinoma [24
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
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Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 Journal of Oncology
tumors cells withwithout chromosomal changes Althoughprevious studies have already reported some tumor specificfusion genes in NPC all of them occur accompanied with therearrangement of chromosomes [13 14]
Herein we carried out RNA sequencing and discovered anovel type of fusion gene trans-splicing chimeric transcriptSEPT7P2-PSPH In addition we performed in vitro studiesto demonstrate that the ability of cell invasion migrationproliferation colony formation and transforming were acti-vated when the expression of the transcript was decreasedindicating that SEPT7P2-PSPH act as the tumor suppressorgene role in NPC tumorigenesis
2 Materials and Methods
21 Primary Tumors and Pair-End RNA Sequencing Tenformalin fixed paraffin embedded (FFPE) NPC samples wereretrieved from the Department of Pathology of Sun Yat-senUniversity Cancer Center (SYSUCCGuangzhou China) Alltumor samples were reviewed by two experienced patholo-gists Total RNA were extracted from ten FFPE tissues withRNeasy FFPE Kit (QIANGEN Hilden Germany) accordingto the manufacturerrsquos instructions for RNA sequencingcDNA libraries were prepared according to the standardprotocol provided by themRNA-seq sample Pre Kit (IlluminaInc California USA) and sequenced (90 nt paired-end) onthe Illumina Hi-seq 2000 To identify fusion genes frompaired-end RNA sequencing the data were analyzed bythe computational pipeline called SOAPfusion which usesclusters of discordant paired-end alignments to inform asplit-read alignment analysis for finding fusion boundaries[15] The UCSC H sapiens reference genome (build hg19)was used for alignments The written informed consent wasobtained from each patient and this study was approvedby Ethics Committee and Institutional Review Board ofSYSUCC
22 Cell Cultures Reverse Transcription and Sanger Sequenc-ing CNE1 and 6-10B used in the study were cultivated inRPMI 1640 (Invitrogen Carlsbad CA USA) supplementedwith 10 FBS (Gibco California USA) in a humidified 5CO2incubator at 37∘C All cultivations involved have been
sustained within 6 months Total RNA was extracted usingTrizol (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos instructions Reverse transcription was sub-sequently performed with PrimeScript RT reagent Kit withgDNA Eraser (TaKaRa Kusatsu Shlga Japan) The productswere employedwith specific primers for Sanger sequencing toconfirm potential fusion transcripts The primerswere shownas follows
SEPT7P2-PSPH 1-sense 51015840-TTACGACTTCTCGGT-CTTCGG-31015840
SEPT7P2-PSPH 1-anti-sense 51015840-CGTCCTCAACGC-CACAGATT-31015840
23 Quantitative RT-PCR Quantitative RT-PCR was per-formed using the ABI 7500 Real-time PCR system (Applied
Biosystems California USA) and SYBR Premix EX Taq(TaKaRa Kusatsu Shlga Japan) All reactions were carriedout in triplicate The housekeeping gene GAPDH was used asnormalization control The relative expression levels of targetgene SEPT7P2-PSPH were calculated by 2minus119862119905 method inwhich Ct = mean Ct SEPT7P2-PSPH ndash mean Ct controlwhere Ct values are the cycle threshold for each sample [16]Specific primers were shown as follows
SEPT7P2-PSPH 2-sense 51015840-ACCTGAGCCTGGGAG-GAAA-31015840
SEPT7P2-PSPH 2-anti-sense 51015840-GTCAACATCAAA-ACACACAGCATC-31015840
24 Immunohistochemistry (IHC) Staining 4120583m thick FFPENPC slides were used to perform IHC staining Firstly thetumor slideswere deparaffinized Antigen retrieval procedurewas then performed in a pressure cooker in 1mM sodium cit-rate (Sangon Biotech Shanghai China) for 15minutes PSPHrabbit polyclonal (Proteintech Wuhan China) was dilutedwith SigalStain antibody diluent (Cell Signaling TechnologyDanvers MA USA) at 1400 for 1 h Universal secondaryantibody (Gene Tech Shanghai China) was applied for 15minutes Subsequently diaminobenzidine was used as chro-mogens and slides were counterstained with haematoxylinbefore mounting PSPH IHC was scored according to thescoring criteria including intensity and percentage as follows0 no staining 1+ faint cytoplasmic reactivity without anybackground staining 2+ moderate cytoplasmic reactivityand 3+ granular cytoplasmic reactivity of strong intensity 00-25 1 25-50 2 50-75 [17]
25 siRNA Transfection Small interfering RNAs which tar-geted the fusion gene (si-SP1 51015840-GUUGUUTTTTCCTCC-CUGG-31015840 si-SP2 51015840-UUGUUTTTTCCTCCCUGGC-31015840)were bought from RIBOBIO Company (Guangzhou China)CNE1 and 6-10B cells were transfected with oligonucleotides(20 nmolL) using the LipofectamineRNAiMAX transfectionreagent (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos instructions
26WesternBlotting Cell lysiswas carried out in RIPAbufferincluding protease inhibitor and phosphatase inhibitor cock-tails (Biostool Shanghai China) The protein extracts wereisolated from the same amounts using 12 SDS-PAGE gelsAfter that all the abstracts were transferred to polyvinylidenefluoride membranes (Merck Millipore Billerica MA USA)Subsequently the membranes were incubated with rabbitpolyclonal anti-PSPH antibody 11000 (Proteintech WuhanChina) And then the incubation was performed with goatanti-rabbit IgG antibody 15000 (Abcam Cambridge UK)Anti-120573-actin antibody (Affinity Ancaster Canada) was actedas a protein control
27 Cell Proliferation Assay Cell proliferation was evaluatedby conducting CCK-8 assays 1000 cells were seeded per wellin 96-well plates in triplicate After incubation with 10uLof CCK8 agent (Dojindo Kumamoto Japan) for 3 hours in
Journal of Oncology 3
the dark in an incubator at 37∘C the absorbance value wasmeasured at 450 nm every 12 hours
28 Boyden Chamber Assays Invasion and migration abili-ties were examined after the siRNA of SEPT7P2-PSPH trans-fection Boyden chambers (Corning New York USA) with 8120583m inserts equipped with (invasion) or without (migration)Matrigel were placed in the 24-well plate A total of 5 times 104cells were suspended in 200 120583L RPMI 1640 without serumin the upper chambers and cultured at 37∘C for 24 hoursThe cells which went through the inserts were stained with05 crystal (Sangon Shanghai China) Then the numberof cells per field of view was counted under phase-contrastmicroscopy
29 Colony Formation Assay 500 transfected cells wereplated in six-well plates and cultured for 7 or 12 daysAll the colonies were fixed with 4 paraformaldehyde andstained with 05 crystal violet (Sangon Shanghai China)for quantification All the experiments were performed intriplicate
210 Anchorage-Independent Assay In the 6-well plate thebottom layers were the mixture of equivalent volumes of12 agarose (Sangon Shanghai China) in PBS and mediumcontaining 20 FBS 5000 cells were suspended in the upperlayer containing 1ml 04 soft agarose
211 Statistical Analysis All statistical analyses were per-formed using SPSS 160 software (IBM Armonk NY USA)and GraphPad Prism version 50 (GraphPad Software) Thedata are presented as the mean plusmn SEM from at leastthree independent experiments Two-tailed Studentrsquos t-testswere conducted to compare the difference between groupsProgression-free survival (PFS) and overall survival (OS)were calculated using the Kaplan-Meier method and the sur-vival curves were compared with the log-rank test methodAll tests were two-sided and P lt 005 was considered asstatistically significant
3 Results
31 Novel Fusion Genes Discovered in NPC We found 60transcripts of fusion genes using RNA sequencing including28 inter- and 32 intrachromosomal fusions from ten FFPENPC tissues (Table 1) Paired-end reads featured by twouncorrelated genes or spanned exon-exon junctions werenominated as putative chimeric genes (Figures 1(a) and 1(b))Fusion genes recurrently occurred in more than one samplewere selected for subsequent investigation A novel fusiongene named SEPT7P2-PSPH was discovered in 5 out of10 samples and located on chromosome 7 (Figure 1(b)) Tovalidate this newly found transcript we designed specificprimers matching the region that contained the fusion pointat the mRNA level The mRNA level of this fusion gene wasvalidated using Sanger sequencing in the 5 samples previouslyand consistent with that identified by RNA sequencing(Figure 1(c))
The transcript was formed with the exon 1 of SEPT7P2and the exon 4 of PSPH The practical distance betweenthe two exons are 102 Mb on chromosome 7 To estimateif there existed a genomic DNA rearrangement with thechimeric RNA we designed 30 primer pairs to amplify theentire genome sequences between exon 1 and exon 2 ofSEPT7P2 as well as the genome sequences between exon 3and exon 4 of PSPH (Supplementary Table 1 and Figure 1(d))However all the segments were amplified by PCR indirectlyindicating the fact that chromosomal arrangementwas absent(Figure 1(e))
32 The Expression of the Fusion Genes in Cell Lines andTissues In addition we have investigated several tumor celllines including Hela Hep3B NP69 CNE1 6-10B SUNE2CNE2 HONE1 and 5-8F All the cell lines were confirmed toharbor the fusion gene (Figure 2(a)) Interestingly we foundthat the relative expression of SEPT7P2-PSPH was higherin the cohort of nasopharyngitis than that in NPC patients(Figure 2(b) P lt 005) Collectively these results indicatedthat the newly found transcript was not peculiar to tumorcells Analogous studies have been published to demonstratethe noncanonical chimeric transcript [10ndash12] Simultane-ously we performed western blotting assay to discover ifthere would be a new type of fusion protein accompanied bythe transcript Anti-PSPH polyclonal antibody was appliedin the nine tumor cell lines It was shown that the putativefusion protein did not exist and only the protein of full-lengthPSPH was confirmed by western blot with the band at 25 kDa(Figure 2(c))
33 Knocking Down SEPT7P2ndashPSPH Upregulated the Levelof PSPH Protein and Promoted Cell Proliferation and Migra-tion We carried out in vitro experiments to confirm thepotential biologic function of the SEPT7P2-PSPH transcriptNPC cell lines CNE1 and 6-10B were selected to explorethe effect of SEPT7P2-PSPH on NPC tumorigenesis Whencompared with the negative control group the expressionof the chimeric transcript substantially decreased after twospecifically designed siRNAs siSP1 and siSP2 were tran-siently transfected into the cell lines Also there was nonovel fusion protein synthesized After transfection for 48hours the expression of SEPT7P2-PSPH at mRNA level wasdecreased sharply whereas the expression of PSPH proteinwas upregulated in both cell lines (Figures 2(d)-2(e) andSupplementary Figure 1) This may indirectly show thephenomenon that the chimeric RNA fusion did not producea new fusion protein
However the expression level of down-stream genersquosprotein was reversely influenced by the occurrence of thefusion between SEPT7P2 and PSPH We subsequently per-formed cell proliferation assays to explore the effects ofthe fusion gene on the growth of CNE-1 and 6-10B Thecells transfected with si-SEPT7P2ndashPSPH-1 (siSP1) and si-SEPT7P2ndashPSPH-2 (siSP1) grew faster than those with neg-ative control (Figure 3(a) P lt 005) Low expression ofSEPT7P2-PSPH may contribute to tumor cell proliferationFor investigating its influence on the transforming ability of
4 Journal of Oncology
(a)
(b)
SEPT7P2 PSPH
(c)
SEPT7P2
PSPH
Exon 1 Exon 2
Exon 3 Exon 4
(d)
SEPT7P2
PSPH
(e)
Figure 1 RNA sequencing was used to discover SEPT7P2-PSPH fusion gene in NPC (a) Detection of SEPT7P2-PSPH fusion gene via RNAsequencing The reads are aligned across the junction of the predicted fusion transcripts (b) Circos plot of SEPT7P2-PSPH fusion generevealed the fusion existing in chromosome 7 (c)The fusion breakpoint was verified using Sanger sequencing (d) A schematic illustration ofthe amplified regions of fusion gene with exons 1-2 of SEPT7P2 and exons 3-4 of PSPH (e)The sequences between exons 1 and 2 of SEPT7P2and the sequences between exons 3 and 4 of PSPH were all amplified by RTndashPCR
Journal of Oncology 5
Table 1 60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues
up gene up chr down gene down chr Fusion TypeIGKC chr2 CALR chr19 INTERCHR-DSIGKJ5 chr2 CALR chr19 INTERCHR-DSMALAT1 chr11 RFWD2 chr1 INTERCHR-DSMRPS18A chr6 C5orf25 chr5 INTERCHR-DSRN7SL1 chr14 IGKC chr2 INTERCHR-DSRN7SL1 chr14 IGKJ5 chr2 INTERCHR-DSRNU6-1 chr15 SNORD3A chr17 INTERCHR-DSRNU6-33 chr4 SNORD3A chr17 INTERCHR-DSRNU6-42 chr3 RPS3 chr11 INTERCHR-DSRNU6-42 chr3 SNORD15A chr11 INTERCHR-DSRNU6-42 chr3 SNORD3A chr17 INTERCHR-DSSAMD12 chr8 ATP10D chr4 INTERCHR-DSATP1B2 chr17 HSD17B12 chr11 INTERCHR-SSE2F4 chr16 RPL14 chr3 INTERCHR-SSEFCAB4A chr11 RN7SK chr6 INTERCHR-SSIGHG1 chr14 NINJ1 chr9 INTERCHR-SSIGKC chr2 CSNK2A2 chr16 INTERCHR-SSIGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SSMIDN chr19 ALMS1 chr2 INTERCHR-SSPDE4B chr1 PPP6R3 chr11 INTERCHR-SSPOU2AF1 chr11 PARP11 chr12 INTERCHR-SSRNU6-2 chr10 SNORD3A chr17 INTERCHR-SSRNU6-36 chr12 SNORD3A chr17 INTERCHR-SSRNU6-42 chr3 SNHG12 chr1 INTERCHR-SSRPL14 chr3 GLS chr2 INTERCHR-SSSLC7A5P2 chr16 PHC3 chr3 INTERCHR-SSSMARCA2 chr9 RPL14 chr3 INTERCHR-SSZNF827 chr4 ZNF318 chr6 INTERCHR-SSADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAPAP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAPC12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAPCOL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAPCTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAPCTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAPEEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAPJAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAPKIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAPLSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAPMAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAPMRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAPNPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAPPIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAPPOLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAPPPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAPPROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAPPSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAPRRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAPSTYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAPTTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAPVMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP
6 Journal of Oncology
Table 1 Continued
up gene up chr down gene down chr Fusion TypeWDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAPWNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAPEEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAPHACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAPIRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAPTMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAPRMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAPFGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGOSEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGOSRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGOchr chromosome
NPC cells we conducted both colony formation assay andanchorage-independent growth assay Strikingly CNE1 and6-10B cells with siSP1 and siSP2 formed larger colonies thanthe negative control groups which was reversely correlatedwith the expression of the chimeric RNA (Figures 3(b) and3(c) P lt 005) To evaluate its biological function withmigration and invasion Boyden chamber assays in vitrohave been conducted As shown in Figure 3(d) knock-ing down SEPT7P2-PSPH with siSP1 and siSP2 markedlyenhanced the migration and invasion of NPC cells (P lt005)
34 PSPH Expression and Prognosis of NPC Patients Toinvestigate the expression status of PSPH in NPC weperformed IHC experiment for PSPH in 72 FFPE NPCspecimens (Figure 4) The result revealed that the patientswith high PSPH expression showed a tendency with shorterPFS and OS in NPC patients though there was no statisticallysignificant (Figures 5(a) and 5(b) P = 0205 P = 0106)
4 Discussion
The present study is the first to identify trans-splicingchimeric transcript SEPT7P2-PSPH without chromosomalrearrangement in NPC patients Knocking down of this tran-scriptmay promote cell proliferation andmetastasisinvasionpossibly due to the upregulation expression of the down-stream gene PSPH
Traditionally chromosome rearrangements can generatefusion genes which are in an intimate relationship withtumor carcinogenesis To date it makes possible to discovermore and more valuable fusion genes acting as a crucialregulator in cancer formation maintenance and evolutionPrevious studies have shown that chimeric RNAs in normaland cancer cells can be generated by intergenic splicing inthe absence of chromosome rearrangements [7ndash9 18 19]This kind of fusion gene can be used as ldquononcanonicalchimerasrdquo It has been reported that there are two meth-ods for ldquononcanonical chimerasrdquo formation involving cis-splicing and trans-splicing of adjacent genes [10] Cis-splicing
fusions tend to occur between two genes that are locatedwithin 30kb in the same chromosome Trans-splicing canbe classified into two types intragenic trans-splicing takingplace between two pre-mRNAs transcribed from the samegenome loci and intergenic trans-splicing that is from twodifferent genome loci SEPT7P2-PSPH can be classified asintergenic trans-splicing located on chromosome 7 alignedwith the fact that the up- and downstream genes are 102Mbdistance
Furthermore we found that metastasisinvasion andproliferation of the tumor cells were inversely enhanced afterthe transcript had been knocked down This was differentfrom previously analogous studies [14 20] The westernblot result showed that there was no newly synthetic fusionprotein accompanied with the chimeric RNA Only the PSPHprotein was translated from the downstream gene PSPHand was overexpressed after the chimeric RNA had beenknocked downThe sequence of the chimeric RNA indicatedthat both of the two genesrsquo start codons were retained afterthe occurrence of the fusion one in exon 1 of SEPT7P2and another in exon 4 of PSPH It seemed that the startcodon in exon 1 of SEPT7P2 depressed the transcriptionof PSPH The reason why fusion genes have the ability ofoncogenesis can be illuminated that the new synthesizedfusion products possess the tumorigenic function Anotherreason is that the preoncogene could be upregulated afterconnected to a stronger promoter or the antioncogenecould be downregulated after fused with a weaker promoter[21]
SEPT7P2 is a pseudogene which has been proved tobe related to SEPT7 and expressed in all tissue types [22]playing a role in multiple biological processes includingvesicle trafficking apoptosis remodeling of the cytoskeletonneurodegeneration and neoplasia
Some researchers have reported that abnormal expressionof PSPH is strongly relevant to the hepatocellular carcinomapatientsrsquo mortality as well as the critical importance oncMyc-induced cancer progression both in vitro and in vivo[23] The level of PSPH has the relationship with breastcancer and lacrimal gland adenoid cystic carcinoma [24
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
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Hindawiwwwhindawicom Volume 2013
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Oncology 3
the dark in an incubator at 37∘C the absorbance value wasmeasured at 450 nm every 12 hours
28 Boyden Chamber Assays Invasion and migration abili-ties were examined after the siRNA of SEPT7P2-PSPH trans-fection Boyden chambers (Corning New York USA) with 8120583m inserts equipped with (invasion) or without (migration)Matrigel were placed in the 24-well plate A total of 5 times 104cells were suspended in 200 120583L RPMI 1640 without serumin the upper chambers and cultured at 37∘C for 24 hoursThe cells which went through the inserts were stained with05 crystal (Sangon Shanghai China) Then the numberof cells per field of view was counted under phase-contrastmicroscopy
29 Colony Formation Assay 500 transfected cells wereplated in six-well plates and cultured for 7 or 12 daysAll the colonies were fixed with 4 paraformaldehyde andstained with 05 crystal violet (Sangon Shanghai China)for quantification All the experiments were performed intriplicate
210 Anchorage-Independent Assay In the 6-well plate thebottom layers were the mixture of equivalent volumes of12 agarose (Sangon Shanghai China) in PBS and mediumcontaining 20 FBS 5000 cells were suspended in the upperlayer containing 1ml 04 soft agarose
211 Statistical Analysis All statistical analyses were per-formed using SPSS 160 software (IBM Armonk NY USA)and GraphPad Prism version 50 (GraphPad Software) Thedata are presented as the mean plusmn SEM from at leastthree independent experiments Two-tailed Studentrsquos t-testswere conducted to compare the difference between groupsProgression-free survival (PFS) and overall survival (OS)were calculated using the Kaplan-Meier method and the sur-vival curves were compared with the log-rank test methodAll tests were two-sided and P lt 005 was considered asstatistically significant
3 Results
31 Novel Fusion Genes Discovered in NPC We found 60transcripts of fusion genes using RNA sequencing including28 inter- and 32 intrachromosomal fusions from ten FFPENPC tissues (Table 1) Paired-end reads featured by twouncorrelated genes or spanned exon-exon junctions werenominated as putative chimeric genes (Figures 1(a) and 1(b))Fusion genes recurrently occurred in more than one samplewere selected for subsequent investigation A novel fusiongene named SEPT7P2-PSPH was discovered in 5 out of10 samples and located on chromosome 7 (Figure 1(b)) Tovalidate this newly found transcript we designed specificprimers matching the region that contained the fusion pointat the mRNA level The mRNA level of this fusion gene wasvalidated using Sanger sequencing in the 5 samples previouslyand consistent with that identified by RNA sequencing(Figure 1(c))
The transcript was formed with the exon 1 of SEPT7P2and the exon 4 of PSPH The practical distance betweenthe two exons are 102 Mb on chromosome 7 To estimateif there existed a genomic DNA rearrangement with thechimeric RNA we designed 30 primer pairs to amplify theentire genome sequences between exon 1 and exon 2 ofSEPT7P2 as well as the genome sequences between exon 3and exon 4 of PSPH (Supplementary Table 1 and Figure 1(d))However all the segments were amplified by PCR indirectlyindicating the fact that chromosomal arrangementwas absent(Figure 1(e))
32 The Expression of the Fusion Genes in Cell Lines andTissues In addition we have investigated several tumor celllines including Hela Hep3B NP69 CNE1 6-10B SUNE2CNE2 HONE1 and 5-8F All the cell lines were confirmed toharbor the fusion gene (Figure 2(a)) Interestingly we foundthat the relative expression of SEPT7P2-PSPH was higherin the cohort of nasopharyngitis than that in NPC patients(Figure 2(b) P lt 005) Collectively these results indicatedthat the newly found transcript was not peculiar to tumorcells Analogous studies have been published to demonstratethe noncanonical chimeric transcript [10ndash12] Simultane-ously we performed western blotting assay to discover ifthere would be a new type of fusion protein accompanied bythe transcript Anti-PSPH polyclonal antibody was appliedin the nine tumor cell lines It was shown that the putativefusion protein did not exist and only the protein of full-lengthPSPH was confirmed by western blot with the band at 25 kDa(Figure 2(c))
33 Knocking Down SEPT7P2ndashPSPH Upregulated the Levelof PSPH Protein and Promoted Cell Proliferation and Migra-tion We carried out in vitro experiments to confirm thepotential biologic function of the SEPT7P2-PSPH transcriptNPC cell lines CNE1 and 6-10B were selected to explorethe effect of SEPT7P2-PSPH on NPC tumorigenesis Whencompared with the negative control group the expressionof the chimeric transcript substantially decreased after twospecifically designed siRNAs siSP1 and siSP2 were tran-siently transfected into the cell lines Also there was nonovel fusion protein synthesized After transfection for 48hours the expression of SEPT7P2-PSPH at mRNA level wasdecreased sharply whereas the expression of PSPH proteinwas upregulated in both cell lines (Figures 2(d)-2(e) andSupplementary Figure 1) This may indirectly show thephenomenon that the chimeric RNA fusion did not producea new fusion protein
However the expression level of down-stream genersquosprotein was reversely influenced by the occurrence of thefusion between SEPT7P2 and PSPH We subsequently per-formed cell proliferation assays to explore the effects ofthe fusion gene on the growth of CNE-1 and 6-10B Thecells transfected with si-SEPT7P2ndashPSPH-1 (siSP1) and si-SEPT7P2ndashPSPH-2 (siSP1) grew faster than those with neg-ative control (Figure 3(a) P lt 005) Low expression ofSEPT7P2-PSPH may contribute to tumor cell proliferationFor investigating its influence on the transforming ability of
4 Journal of Oncology
(a)
(b)
SEPT7P2 PSPH
(c)
SEPT7P2
PSPH
Exon 1 Exon 2
Exon 3 Exon 4
(d)
SEPT7P2
PSPH
(e)
Figure 1 RNA sequencing was used to discover SEPT7P2-PSPH fusion gene in NPC (a) Detection of SEPT7P2-PSPH fusion gene via RNAsequencing The reads are aligned across the junction of the predicted fusion transcripts (b) Circos plot of SEPT7P2-PSPH fusion generevealed the fusion existing in chromosome 7 (c)The fusion breakpoint was verified using Sanger sequencing (d) A schematic illustration ofthe amplified regions of fusion gene with exons 1-2 of SEPT7P2 and exons 3-4 of PSPH (e)The sequences between exons 1 and 2 of SEPT7P2and the sequences between exons 3 and 4 of PSPH were all amplified by RTndashPCR
Journal of Oncology 5
Table 1 60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues
up gene up chr down gene down chr Fusion TypeIGKC chr2 CALR chr19 INTERCHR-DSIGKJ5 chr2 CALR chr19 INTERCHR-DSMALAT1 chr11 RFWD2 chr1 INTERCHR-DSMRPS18A chr6 C5orf25 chr5 INTERCHR-DSRN7SL1 chr14 IGKC chr2 INTERCHR-DSRN7SL1 chr14 IGKJ5 chr2 INTERCHR-DSRNU6-1 chr15 SNORD3A chr17 INTERCHR-DSRNU6-33 chr4 SNORD3A chr17 INTERCHR-DSRNU6-42 chr3 RPS3 chr11 INTERCHR-DSRNU6-42 chr3 SNORD15A chr11 INTERCHR-DSRNU6-42 chr3 SNORD3A chr17 INTERCHR-DSSAMD12 chr8 ATP10D chr4 INTERCHR-DSATP1B2 chr17 HSD17B12 chr11 INTERCHR-SSE2F4 chr16 RPL14 chr3 INTERCHR-SSEFCAB4A chr11 RN7SK chr6 INTERCHR-SSIGHG1 chr14 NINJ1 chr9 INTERCHR-SSIGKC chr2 CSNK2A2 chr16 INTERCHR-SSIGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SSMIDN chr19 ALMS1 chr2 INTERCHR-SSPDE4B chr1 PPP6R3 chr11 INTERCHR-SSPOU2AF1 chr11 PARP11 chr12 INTERCHR-SSRNU6-2 chr10 SNORD3A chr17 INTERCHR-SSRNU6-36 chr12 SNORD3A chr17 INTERCHR-SSRNU6-42 chr3 SNHG12 chr1 INTERCHR-SSRPL14 chr3 GLS chr2 INTERCHR-SSSLC7A5P2 chr16 PHC3 chr3 INTERCHR-SSSMARCA2 chr9 RPL14 chr3 INTERCHR-SSZNF827 chr4 ZNF318 chr6 INTERCHR-SSADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAPAP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAPC12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAPCOL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAPCTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAPCTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAPEEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAPJAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAPKIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAPLSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAPMAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAPMRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAPNPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAPPIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAPPOLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAPPPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAPPROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAPPSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAPRRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAPSTYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAPTTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAPVMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP
6 Journal of Oncology
Table 1 Continued
up gene up chr down gene down chr Fusion TypeWDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAPWNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAPEEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAPHACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAPIRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAPTMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAPRMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAPFGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGOSEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGOSRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGOchr chromosome
NPC cells we conducted both colony formation assay andanchorage-independent growth assay Strikingly CNE1 and6-10B cells with siSP1 and siSP2 formed larger colonies thanthe negative control groups which was reversely correlatedwith the expression of the chimeric RNA (Figures 3(b) and3(c) P lt 005) To evaluate its biological function withmigration and invasion Boyden chamber assays in vitrohave been conducted As shown in Figure 3(d) knock-ing down SEPT7P2-PSPH with siSP1 and siSP2 markedlyenhanced the migration and invasion of NPC cells (P lt005)
34 PSPH Expression and Prognosis of NPC Patients Toinvestigate the expression status of PSPH in NPC weperformed IHC experiment for PSPH in 72 FFPE NPCspecimens (Figure 4) The result revealed that the patientswith high PSPH expression showed a tendency with shorterPFS and OS in NPC patients though there was no statisticallysignificant (Figures 5(a) and 5(b) P = 0205 P = 0106)
4 Discussion
The present study is the first to identify trans-splicingchimeric transcript SEPT7P2-PSPH without chromosomalrearrangement in NPC patients Knocking down of this tran-scriptmay promote cell proliferation andmetastasisinvasionpossibly due to the upregulation expression of the down-stream gene PSPH
Traditionally chromosome rearrangements can generatefusion genes which are in an intimate relationship withtumor carcinogenesis To date it makes possible to discovermore and more valuable fusion genes acting as a crucialregulator in cancer formation maintenance and evolutionPrevious studies have shown that chimeric RNAs in normaland cancer cells can be generated by intergenic splicing inthe absence of chromosome rearrangements [7ndash9 18 19]This kind of fusion gene can be used as ldquononcanonicalchimerasrdquo It has been reported that there are two meth-ods for ldquononcanonical chimerasrdquo formation involving cis-splicing and trans-splicing of adjacent genes [10] Cis-splicing
fusions tend to occur between two genes that are locatedwithin 30kb in the same chromosome Trans-splicing canbe classified into two types intragenic trans-splicing takingplace between two pre-mRNAs transcribed from the samegenome loci and intergenic trans-splicing that is from twodifferent genome loci SEPT7P2-PSPH can be classified asintergenic trans-splicing located on chromosome 7 alignedwith the fact that the up- and downstream genes are 102Mbdistance
Furthermore we found that metastasisinvasion andproliferation of the tumor cells were inversely enhanced afterthe transcript had been knocked down This was differentfrom previously analogous studies [14 20] The westernblot result showed that there was no newly synthetic fusionprotein accompanied with the chimeric RNA Only the PSPHprotein was translated from the downstream gene PSPHand was overexpressed after the chimeric RNA had beenknocked downThe sequence of the chimeric RNA indicatedthat both of the two genesrsquo start codons were retained afterthe occurrence of the fusion one in exon 1 of SEPT7P2and another in exon 4 of PSPH It seemed that the startcodon in exon 1 of SEPT7P2 depressed the transcriptionof PSPH The reason why fusion genes have the ability ofoncogenesis can be illuminated that the new synthesizedfusion products possess the tumorigenic function Anotherreason is that the preoncogene could be upregulated afterconnected to a stronger promoter or the antioncogenecould be downregulated after fused with a weaker promoter[21]
SEPT7P2 is a pseudogene which has been proved tobe related to SEPT7 and expressed in all tissue types [22]playing a role in multiple biological processes includingvesicle trafficking apoptosis remodeling of the cytoskeletonneurodegeneration and neoplasia
Some researchers have reported that abnormal expressionof PSPH is strongly relevant to the hepatocellular carcinomapatientsrsquo mortality as well as the critical importance oncMyc-induced cancer progression both in vitro and in vivo[23] The level of PSPH has the relationship with breastcancer and lacrimal gland adenoid cystic carcinoma [24
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
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Hindawiwwwhindawicom Volume 2013
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 Journal of Oncology
(a)
(b)
SEPT7P2 PSPH
(c)
SEPT7P2
PSPH
Exon 1 Exon 2
Exon 3 Exon 4
(d)
SEPT7P2
PSPH
(e)
Figure 1 RNA sequencing was used to discover SEPT7P2-PSPH fusion gene in NPC (a) Detection of SEPT7P2-PSPH fusion gene via RNAsequencing The reads are aligned across the junction of the predicted fusion transcripts (b) Circos plot of SEPT7P2-PSPH fusion generevealed the fusion existing in chromosome 7 (c)The fusion breakpoint was verified using Sanger sequencing (d) A schematic illustration ofthe amplified regions of fusion gene with exons 1-2 of SEPT7P2 and exons 3-4 of PSPH (e)The sequences between exons 1 and 2 of SEPT7P2and the sequences between exons 3 and 4 of PSPH were all amplified by RTndashPCR
Journal of Oncology 5
Table 1 60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues
up gene up chr down gene down chr Fusion TypeIGKC chr2 CALR chr19 INTERCHR-DSIGKJ5 chr2 CALR chr19 INTERCHR-DSMALAT1 chr11 RFWD2 chr1 INTERCHR-DSMRPS18A chr6 C5orf25 chr5 INTERCHR-DSRN7SL1 chr14 IGKC chr2 INTERCHR-DSRN7SL1 chr14 IGKJ5 chr2 INTERCHR-DSRNU6-1 chr15 SNORD3A chr17 INTERCHR-DSRNU6-33 chr4 SNORD3A chr17 INTERCHR-DSRNU6-42 chr3 RPS3 chr11 INTERCHR-DSRNU6-42 chr3 SNORD15A chr11 INTERCHR-DSRNU6-42 chr3 SNORD3A chr17 INTERCHR-DSSAMD12 chr8 ATP10D chr4 INTERCHR-DSATP1B2 chr17 HSD17B12 chr11 INTERCHR-SSE2F4 chr16 RPL14 chr3 INTERCHR-SSEFCAB4A chr11 RN7SK chr6 INTERCHR-SSIGHG1 chr14 NINJ1 chr9 INTERCHR-SSIGKC chr2 CSNK2A2 chr16 INTERCHR-SSIGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SSMIDN chr19 ALMS1 chr2 INTERCHR-SSPDE4B chr1 PPP6R3 chr11 INTERCHR-SSPOU2AF1 chr11 PARP11 chr12 INTERCHR-SSRNU6-2 chr10 SNORD3A chr17 INTERCHR-SSRNU6-36 chr12 SNORD3A chr17 INTERCHR-SSRNU6-42 chr3 SNHG12 chr1 INTERCHR-SSRPL14 chr3 GLS chr2 INTERCHR-SSSLC7A5P2 chr16 PHC3 chr3 INTERCHR-SSSMARCA2 chr9 RPL14 chr3 INTERCHR-SSZNF827 chr4 ZNF318 chr6 INTERCHR-SSADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAPAP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAPC12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAPCOL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAPCTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAPCTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAPEEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAPJAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAPKIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAPLSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAPMAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAPMRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAPNPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAPPIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAPPOLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAPPPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAPPROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAPPSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAPRRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAPSTYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAPTTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAPVMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP
6 Journal of Oncology
Table 1 Continued
up gene up chr down gene down chr Fusion TypeWDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAPWNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAPEEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAPHACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAPIRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAPTMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAPRMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAPFGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGOSEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGOSRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGOchr chromosome
NPC cells we conducted both colony formation assay andanchorage-independent growth assay Strikingly CNE1 and6-10B cells with siSP1 and siSP2 formed larger colonies thanthe negative control groups which was reversely correlatedwith the expression of the chimeric RNA (Figures 3(b) and3(c) P lt 005) To evaluate its biological function withmigration and invasion Boyden chamber assays in vitrohave been conducted As shown in Figure 3(d) knock-ing down SEPT7P2-PSPH with siSP1 and siSP2 markedlyenhanced the migration and invasion of NPC cells (P lt005)
34 PSPH Expression and Prognosis of NPC Patients Toinvestigate the expression status of PSPH in NPC weperformed IHC experiment for PSPH in 72 FFPE NPCspecimens (Figure 4) The result revealed that the patientswith high PSPH expression showed a tendency with shorterPFS and OS in NPC patients though there was no statisticallysignificant (Figures 5(a) and 5(b) P = 0205 P = 0106)
4 Discussion
The present study is the first to identify trans-splicingchimeric transcript SEPT7P2-PSPH without chromosomalrearrangement in NPC patients Knocking down of this tran-scriptmay promote cell proliferation andmetastasisinvasionpossibly due to the upregulation expression of the down-stream gene PSPH
Traditionally chromosome rearrangements can generatefusion genes which are in an intimate relationship withtumor carcinogenesis To date it makes possible to discovermore and more valuable fusion genes acting as a crucialregulator in cancer formation maintenance and evolutionPrevious studies have shown that chimeric RNAs in normaland cancer cells can be generated by intergenic splicing inthe absence of chromosome rearrangements [7ndash9 18 19]This kind of fusion gene can be used as ldquononcanonicalchimerasrdquo It has been reported that there are two meth-ods for ldquononcanonical chimerasrdquo formation involving cis-splicing and trans-splicing of adjacent genes [10] Cis-splicing
fusions tend to occur between two genes that are locatedwithin 30kb in the same chromosome Trans-splicing canbe classified into two types intragenic trans-splicing takingplace between two pre-mRNAs transcribed from the samegenome loci and intergenic trans-splicing that is from twodifferent genome loci SEPT7P2-PSPH can be classified asintergenic trans-splicing located on chromosome 7 alignedwith the fact that the up- and downstream genes are 102Mbdistance
Furthermore we found that metastasisinvasion andproliferation of the tumor cells were inversely enhanced afterthe transcript had been knocked down This was differentfrom previously analogous studies [14 20] The westernblot result showed that there was no newly synthetic fusionprotein accompanied with the chimeric RNA Only the PSPHprotein was translated from the downstream gene PSPHand was overexpressed after the chimeric RNA had beenknocked downThe sequence of the chimeric RNA indicatedthat both of the two genesrsquo start codons were retained afterthe occurrence of the fusion one in exon 1 of SEPT7P2and another in exon 4 of PSPH It seemed that the startcodon in exon 1 of SEPT7P2 depressed the transcriptionof PSPH The reason why fusion genes have the ability ofoncogenesis can be illuminated that the new synthesizedfusion products possess the tumorigenic function Anotherreason is that the preoncogene could be upregulated afterconnected to a stronger promoter or the antioncogenecould be downregulated after fused with a weaker promoter[21]
SEPT7P2 is a pseudogene which has been proved tobe related to SEPT7 and expressed in all tissue types [22]playing a role in multiple biological processes includingvesicle trafficking apoptosis remodeling of the cytoskeletonneurodegeneration and neoplasia
Some researchers have reported that abnormal expressionof PSPH is strongly relevant to the hepatocellular carcinomapatientsrsquo mortality as well as the critical importance oncMyc-induced cancer progression both in vitro and in vivo[23] The level of PSPH has the relationship with breastcancer and lacrimal gland adenoid cystic carcinoma [24
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
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of
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Hindawiwwwhindawicom Volume 2018
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Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Oncology 5
Table 1 60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues
up gene up chr down gene down chr Fusion TypeIGKC chr2 CALR chr19 INTERCHR-DSIGKJ5 chr2 CALR chr19 INTERCHR-DSMALAT1 chr11 RFWD2 chr1 INTERCHR-DSMRPS18A chr6 C5orf25 chr5 INTERCHR-DSRN7SL1 chr14 IGKC chr2 INTERCHR-DSRN7SL1 chr14 IGKJ5 chr2 INTERCHR-DSRNU6-1 chr15 SNORD3A chr17 INTERCHR-DSRNU6-33 chr4 SNORD3A chr17 INTERCHR-DSRNU6-42 chr3 RPS3 chr11 INTERCHR-DSRNU6-42 chr3 SNORD15A chr11 INTERCHR-DSRNU6-42 chr3 SNORD3A chr17 INTERCHR-DSSAMD12 chr8 ATP10D chr4 INTERCHR-DSATP1B2 chr17 HSD17B12 chr11 INTERCHR-SSE2F4 chr16 RPL14 chr3 INTERCHR-SSEFCAB4A chr11 RN7SK chr6 INTERCHR-SSIGHG1 chr14 NINJ1 chr9 INTERCHR-SSIGKC chr2 CSNK2A2 chr16 INTERCHR-SSIGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SSMIDN chr19 ALMS1 chr2 INTERCHR-SSPDE4B chr1 PPP6R3 chr11 INTERCHR-SSPOU2AF1 chr11 PARP11 chr12 INTERCHR-SSRNU6-2 chr10 SNORD3A chr17 INTERCHR-SSRNU6-36 chr12 SNORD3A chr17 INTERCHR-SSRNU6-42 chr3 SNHG12 chr1 INTERCHR-SSRPL14 chr3 GLS chr2 INTERCHR-SSSLC7A5P2 chr16 PHC3 chr3 INTERCHR-SSSMARCA2 chr9 RPL14 chr3 INTERCHR-SSZNF827 chr4 ZNF318 chr6 INTERCHR-SSADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAPAP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAPC12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAPCOL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAPCTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAPCTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAPEEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAPJAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAPKIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAPLSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAPMAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAPMRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAPNPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAPPIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAPPOLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAPPPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAPPROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAPPSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAPRRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAPSTYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAPTTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAPVMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP
6 Journal of Oncology
Table 1 Continued
up gene up chr down gene down chr Fusion TypeWDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAPWNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAPEEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAPHACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAPIRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAPTMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAPRMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAPFGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGOSEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGOSRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGOchr chromosome
NPC cells we conducted both colony formation assay andanchorage-independent growth assay Strikingly CNE1 and6-10B cells with siSP1 and siSP2 formed larger colonies thanthe negative control groups which was reversely correlatedwith the expression of the chimeric RNA (Figures 3(b) and3(c) P lt 005) To evaluate its biological function withmigration and invasion Boyden chamber assays in vitrohave been conducted As shown in Figure 3(d) knock-ing down SEPT7P2-PSPH with siSP1 and siSP2 markedlyenhanced the migration and invasion of NPC cells (P lt005)
34 PSPH Expression and Prognosis of NPC Patients Toinvestigate the expression status of PSPH in NPC weperformed IHC experiment for PSPH in 72 FFPE NPCspecimens (Figure 4) The result revealed that the patientswith high PSPH expression showed a tendency with shorterPFS and OS in NPC patients though there was no statisticallysignificant (Figures 5(a) and 5(b) P = 0205 P = 0106)
4 Discussion
The present study is the first to identify trans-splicingchimeric transcript SEPT7P2-PSPH without chromosomalrearrangement in NPC patients Knocking down of this tran-scriptmay promote cell proliferation andmetastasisinvasionpossibly due to the upregulation expression of the down-stream gene PSPH
Traditionally chromosome rearrangements can generatefusion genes which are in an intimate relationship withtumor carcinogenesis To date it makes possible to discovermore and more valuable fusion genes acting as a crucialregulator in cancer formation maintenance and evolutionPrevious studies have shown that chimeric RNAs in normaland cancer cells can be generated by intergenic splicing inthe absence of chromosome rearrangements [7ndash9 18 19]This kind of fusion gene can be used as ldquononcanonicalchimerasrdquo It has been reported that there are two meth-ods for ldquononcanonical chimerasrdquo formation involving cis-splicing and trans-splicing of adjacent genes [10] Cis-splicing
fusions tend to occur between two genes that are locatedwithin 30kb in the same chromosome Trans-splicing canbe classified into two types intragenic trans-splicing takingplace between two pre-mRNAs transcribed from the samegenome loci and intergenic trans-splicing that is from twodifferent genome loci SEPT7P2-PSPH can be classified asintergenic trans-splicing located on chromosome 7 alignedwith the fact that the up- and downstream genes are 102Mbdistance
Furthermore we found that metastasisinvasion andproliferation of the tumor cells were inversely enhanced afterthe transcript had been knocked down This was differentfrom previously analogous studies [14 20] The westernblot result showed that there was no newly synthetic fusionprotein accompanied with the chimeric RNA Only the PSPHprotein was translated from the downstream gene PSPHand was overexpressed after the chimeric RNA had beenknocked downThe sequence of the chimeric RNA indicatedthat both of the two genesrsquo start codons were retained afterthe occurrence of the fusion one in exon 1 of SEPT7P2and another in exon 4 of PSPH It seemed that the startcodon in exon 1 of SEPT7P2 depressed the transcriptionof PSPH The reason why fusion genes have the ability ofoncogenesis can be illuminated that the new synthesizedfusion products possess the tumorigenic function Anotherreason is that the preoncogene could be upregulated afterconnected to a stronger promoter or the antioncogenecould be downregulated after fused with a weaker promoter[21]
SEPT7P2 is a pseudogene which has been proved tobe related to SEPT7 and expressed in all tissue types [22]playing a role in multiple biological processes includingvesicle trafficking apoptosis remodeling of the cytoskeletonneurodegeneration and neoplasia
Some researchers have reported that abnormal expressionof PSPH is strongly relevant to the hepatocellular carcinomapatientsrsquo mortality as well as the critical importance oncMyc-induced cancer progression both in vitro and in vivo[23] The level of PSPH has the relationship with breastcancer and lacrimal gland adenoid cystic carcinoma [24
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 Journal of Oncology
Table 1 Continued
up gene up chr down gene down chr Fusion TypeWDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAPWNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAPEEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAPHACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAPIRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAPTMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAPRMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAPFGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGOSEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGOSRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGOchr chromosome
NPC cells we conducted both colony formation assay andanchorage-independent growth assay Strikingly CNE1 and6-10B cells with siSP1 and siSP2 formed larger colonies thanthe negative control groups which was reversely correlatedwith the expression of the chimeric RNA (Figures 3(b) and3(c) P lt 005) To evaluate its biological function withmigration and invasion Boyden chamber assays in vitrohave been conducted As shown in Figure 3(d) knock-ing down SEPT7P2-PSPH with siSP1 and siSP2 markedlyenhanced the migration and invasion of NPC cells (P lt005)
34 PSPH Expression and Prognosis of NPC Patients Toinvestigate the expression status of PSPH in NPC weperformed IHC experiment for PSPH in 72 FFPE NPCspecimens (Figure 4) The result revealed that the patientswith high PSPH expression showed a tendency with shorterPFS and OS in NPC patients though there was no statisticallysignificant (Figures 5(a) and 5(b) P = 0205 P = 0106)
4 Discussion
The present study is the first to identify trans-splicingchimeric transcript SEPT7P2-PSPH without chromosomalrearrangement in NPC patients Knocking down of this tran-scriptmay promote cell proliferation andmetastasisinvasionpossibly due to the upregulation expression of the down-stream gene PSPH
Traditionally chromosome rearrangements can generatefusion genes which are in an intimate relationship withtumor carcinogenesis To date it makes possible to discovermore and more valuable fusion genes acting as a crucialregulator in cancer formation maintenance and evolutionPrevious studies have shown that chimeric RNAs in normaland cancer cells can be generated by intergenic splicing inthe absence of chromosome rearrangements [7ndash9 18 19]This kind of fusion gene can be used as ldquononcanonicalchimerasrdquo It has been reported that there are two meth-ods for ldquononcanonical chimerasrdquo formation involving cis-splicing and trans-splicing of adjacent genes [10] Cis-splicing
fusions tend to occur between two genes that are locatedwithin 30kb in the same chromosome Trans-splicing canbe classified into two types intragenic trans-splicing takingplace between two pre-mRNAs transcribed from the samegenome loci and intergenic trans-splicing that is from twodifferent genome loci SEPT7P2-PSPH can be classified asintergenic trans-splicing located on chromosome 7 alignedwith the fact that the up- and downstream genes are 102Mbdistance
Furthermore we found that metastasisinvasion andproliferation of the tumor cells were inversely enhanced afterthe transcript had been knocked down This was differentfrom previously analogous studies [14 20] The westernblot result showed that there was no newly synthetic fusionprotein accompanied with the chimeric RNA Only the PSPHprotein was translated from the downstream gene PSPHand was overexpressed after the chimeric RNA had beenknocked downThe sequence of the chimeric RNA indicatedthat both of the two genesrsquo start codons were retained afterthe occurrence of the fusion one in exon 1 of SEPT7P2and another in exon 4 of PSPH It seemed that the startcodon in exon 1 of SEPT7P2 depressed the transcriptionof PSPH The reason why fusion genes have the ability ofoncogenesis can be illuminated that the new synthesizedfusion products possess the tumorigenic function Anotherreason is that the preoncogene could be upregulated afterconnected to a stronger promoter or the antioncogenecould be downregulated after fused with a weaker promoter[21]
SEPT7P2 is a pseudogene which has been proved tobe related to SEPT7 and expressed in all tissue types [22]playing a role in multiple biological processes includingvesicle trafficking apoptosis remodeling of the cytoskeletonneurodegeneration and neoplasia
Some researchers have reported that abnormal expressionof PSPH is strongly relevant to the hepatocellular carcinomapatientsrsquo mortality as well as the critical importance oncMyc-induced cancer progression both in vitro and in vivo[23] The level of PSPH has the relationship with breastcancer and lacrimal gland adenoid cystic carcinoma [24
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Oncology 7
SEPT7P2-PSPH
GAPDH
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(a)
0
5
10
15
20
NPC patien
ts
Nasophary
ngitis
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
lowastlowastlowast
(b)
PSPH
ACTIN
CNE1
SUN
E2CN
E2H
ON
E15-
8F
6-10
B
Hel
a
NP6
9H
ep3B
(c)
si-contro
l
si-SE
PT7P2-
PSPH-1
10
05
15
0Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-2 si-
control
0
05
10
15
Relat
ive e
xpre
ssio
n of
SEPT
7P2-
PSPH
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
CNE1 6-10B
(d)
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
CNE1 6-10B
ACTIN
PSPH
si-SE
PT7P2-
PSPH-1si-
control
si-contro
l
si-SE
PT7P2-
PSPH-2
(e)
Figure 2 SEPT7P2-PSPH expression (a) By RTndashPCR the SEPT7P2-PSPH fusion transcripts were verified in nine cell lines (b) The relativeexpression of SEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P lt 005) (c)Full-lengthPSPH proteinwas expressed in nine cell lines bywestern blotting andno relevant chimeric fusion proteinwas detected Expressionof SEPT7P2-PSPH was knocked down by the specific siRNAThe suppression effect was verified by quantitative RT-PCR andwestern blottingrespectively (d e n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)
25] In our study high expression of PSPH was the ten-dency to NPC patientsrsquo poor survival though there was nostatistically significant Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonicand hematopoietic stem cells and neural progenitors of thedeveloping brain [26 27] Furthermore inhibition of PSPHinduced programmed cell death in tonsillar cell cultures [28]In another words PSPH plays an important oncogenic rolein regulating the ability of cell proliferation and apoptosis
Hence we put forward the hypothesis that it could bethe PSPH overexpression induced when the pseudogeneSEPT7P2 and PSPH fuse with each other Recently somestudies demonstrated that trans-splicing can become one ofthe tumor-targeting methods in cancer related treatment inwhich trans-splicing ribozyme may function as a potentialanticancer agent via stimulating anticancer gene activity [29]However such role of the transcript SEPT7P2-PSPH in NPCwarrants the further study
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 Journal of Oncology
0
2
4
6
8
Time (hours)0 12 24 36 48 60 72
Relat
ive c
ell p
rolif
erat
ion
0
2
4
1
3
Relat
ive c
ell p
rolif
erat
ion
0 12 24 36 48 60Time (hours)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowast
CNE1-si-SEPT7P2-PSPH-1
CNE1-si-controlCNE1-si-SEPT7P2-PSPH-2
6-10B-si-SEPT7P2-PSPH-1
6-10B-si-control6-10B-si-SEPT7P2-PSPH-2
(a)
CNE1
si-control
6-10B
si-control
Num
ber o
f col
onie
s
0
50
100
150
0
50
100
150
200
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowastlowast
(b)
CNE1
6-10B
si-control
0
50
100
150
200
250
Num
ber o
f col
onie
s0
50
100
150
200
250
Num
ber o
f col
onie
s
si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-contro
l
si-SE
PT7P2-
PSPH-1
si-SE
PT7P2-
PSPH-2
si-controlsi-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
lowastlowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
CNE1 6-10B
Migration
Invasion
si-control si-SEPT7P2-PSPH-1 si-controlsi-SEPT7P2-
PSPH-2si-SEPT7P2-PSPH-1
si-SEPT7P2-PSPH-2
(d)
Figure 3 Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells (a) CCK8 assays showed that cellproliferation was significantly enhanced after the specific siRNA interfered with the expression of SEPT7P2-PSPH (n = 3 two-tailed Studentrsquost-tests lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formationability of CNE1 and 6-10B cells (n = 3 two-tailed Studentrsquos t-tests lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) (d) Cell migration and invasion assaysrevealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA
5 Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in ourstudy might be a tumor suppressor gene in NPC tumorigen-esis potentially having the role of anticancer activity
Data Availability
The raw data in this paper has been successfully uploadedand locked onto Research Data Deposit with RDD noRDDB2019000527
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Oncology 9
(a) (b)
(c) (d)
Figure 4 Representative IHC staining with negative (a) low (b) moderate (c) high and (d) PSPH expression Scale bar left panel 500120583mright panel 100120583m
0
50
100
0 20 40 60 80Months
Prog
ress
ion-
free
surv
ival
()
Low expressionHigh expression
P=0205
(a)
Months
0
50
100
0 20 40 60 80 100
Low expressionHigh expression
P=0106
Ove
rall
surv
ival
()
(b)
Figure 5 Survival analysis (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low(n=27) versus high (n=45) expression of PSPH through immunohistochemistry (IHC) staining P value was determined by the log-rank test(P gt 005)
Conflicts of Interest
The authors declare no conflicts of interest
Authorsrsquo Contributions
Jing Wang and Guo-Feng Xie are equal contributors
Acknowledgments
This work was supported by The National Key Researchand Development Program of China (2016YFC0902000) andNational Natural Science Foundation of China (8160246881472522) We thank the patients who participated in ourstudy
Supplementary Materials
Supplementary Figure 1 The PSPH expression was verifiedby western blotting after interfering expression of SEPT7P2-PSPH by the two specific siRNA Supplementary Table 1Primer sequences (Supplementary Materials)
References
[1] M Al-Sarraf and M S Reddy ldquoNasopharyngeal carcinomardquoCurrent Treatment Options in Oncology vol 3 no 1 pp 21ndash322002
[2] K W Lo K F To and D P Huang ldquoFocus on nasopharyngealcarcinomardquo Cancer Cell vol 5 no 5 pp 423ndash428 2004
[3] F Mitelman B Johansson and F Mertens ldquoThe impact oftranslocations and gene fusions on cancer causationrdquo NatureReviews Cancer vol 7 no 4 pp 233ndash245 2007
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
10 Journal of Oncology
[4] T Kohno H Ichikawa Y Totoki et al ldquoKIF5B-RET fusions inlung adenocarcinomardquoNature Medicine vol 18 no 3 pp 375ndash377 2012
[5] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009
[6] D S Rickman D Pflueger B Moss et al ldquoSLC45A3-ELK4 is anovel and frequent erythroblast transformation-specific fusiontranscript in prostate cancerrdquoCancer Research vol 69 no 7 pp2737-2738 2009
[7] Y Zhang M Gong H Yuan H G Park H F Friersonand H Li ldquoChimeric transcript generated by cis-splicing ofadjacent genes regulates prostate cancer cell proliferationrdquoCancer Discovery vol 2 no 7 pp 598ndash607 2012
[8] H Li J Wang G Mor and J Sklar ldquoA neoplastic gene fusionmimics trans-splicing of RNAs in normal human cellsrdquo Sciencevol 321 no 5894 pp 1357ndash1361 2008
[9] H Yuan F Qin M Movassagh et al ldquoA chimeric RNAcharacteristic of rhabdomyosarcoma in normal myogenesisprocessrdquo Cancer Discovery vol 3 no 12 pp 1394ndash1403 2013
[10] K Jividen and H Li ldquoChimeric RNAs generated by intergenicsplicing in normal and cancer cellsrdquo Genes Chromosomes andCancer vol 53 no 12 pp 963ndash971 2014
[11] J-H Luo S Liu Z-H Zuo R Chen G C Tseng and Y P YuldquoDiscovery and classification of fusion transcripts in prostatecancer and normal prostate tissuerdquo The American Journal ofPathology vol 185 no 7 pp 1834ndash1845 2015
[12] M Babiceanu F Qin Z Xie et al ldquoRecurrent chimeric fusionRNAs in non-cancer tissues and cellsrdquo Nucleic Acids Researchvol 44 no 6 pp 2859ndash2872 2016
[13] L Yuan Z-H Liu Z-R Lin L-H Xu Q Zhong and M-SZeng ldquoRecurrent FGFR3-TACC3 fusion gene in nasopharyn-geal carcinomardquo Cancer Biology amp Therapy vol 15 no 12 pp1613ndash1621 2014
[14] G T Y Chung RWM Lung A B Y Hui et al ldquoIdentificationof a recurrent transforming UBR5-ZNF423 fusion gene inEBV-associated nasopharyngeal carcinomardquo The Journal ofPathology vol 231 no 2 pp 158ndash167 2013
[15] J Wu W Zhang S Huang et al ldquoSOAPfusion a robustand effective computational fusion discovery tool for RNA-seqreadsrdquo Bioinformatics vol 29 no 23 pp 2971ndash2978 2013
[16] L X Yan X F Huang Q Shao et al ldquoMicroRNA miR-21 overexpression in human breast cancer is associated withadvanced clinical stage lymphnodemetastasis and patient poorprognosisrdquo RNA vol 14 no 11 pp 2348ndash2360 2008
[17] H Y Wang B Y Sun Z H Zhu et al ldquoEight-signature clas-sifier for prediction of nasopharyngeal [corrected] carcinomasurvivalrdquo Journal of Clinical Oncology Official Journal of theAmerican Society of Clinical Oncology vol 29 pp 4516ndash45252011
[18] J Lai M L LehmanM E Dinger et al ldquoA variant of the KLK4gene is expressed as a cis sense-antisense chimeric transcript inprostate cancer cellsrdquo RNA vol 16 no 6 pp 1156ndash1166 2010
[19] TVelusamyN Palanisamy SKalyana-Sundaramet al ldquoRecur-rent reciprocal RNA chimera involving YPEL5 and PPP1CBin chronic lymphocytic leukemiardquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 110 no8 pp 3035ndash3040 2013
[20] D Singh J M Chan P Zoppoli et al ldquoTransforming fusionsof FGFR and TACC genes in human glioblastomardquo Science vol337 no 6099 pp 1231ndash1235 2012
[21] J Zhou J Liao X Zheng and H Shen ldquoChimeric RNAs aspotential biomarkers for tumor diagnosisrdquo BMB Reports vol45 pp 133ndash140 2012
[22] P A Hall K Jung K J Hillan and S E H Russell ldquoExpressionprofiling the human septin gene familyrdquo The Journal of Pathol-ogy vol 206 no 3 pp 269ndash278 2005
[23] L Sun L Song Q Wan et al ldquocMyc-mediated activation ofserine biosynthesis pathway is critical for cancer progressionunder nutrient deprivation conditionsrdquo Cell Research vol 25pp 429ndash444 2015
[24] H M Kim W H Jung and J S Koo ldquoSite-specific metabolicphenotypes inmetastatic breast cancerrdquo Journal of TranslationalMedicine vol 12 2014
[25] J S Koo and J S Yoon ldquoExpression of metabolism-relatedproteins in lacrimal gland adenoid cystic carcinomardquoAmericanJournal of Clinical Pathology vol 143 no 4 pp 584ndash592 2015
[26] D H Geschwind J Ou M C Easterday et al ldquoA geneticanalysis of neural progenitor differentiationrdquo Neuron vol 29no 2 pp 325ndash339 2001
[27] I Nakano J D Dougherty K Kim I Klement D HGeschwind and H I Kornblum ldquoPhosphoserine phosphataseis expressed in the neural stem cell niche and regulates neuralstem and progenitor cell proliferationrdquo Stem Cells vol 25 no 8pp 1975ndash1984 2007
[28] A Khalyfa S A Gharib J Kim et al ldquoTranscriptomic anal-ysis identifies phosphatases as novel targets for adenotonsillarhypertrophy of pediatric obstructive sleep apneardquo AmericanJournal of Respiratory and Critical Care Medicine vol 181 no10 pp 1114ndash1120 2010
[29] S-W Lee and J-S Jeong ldquoUse of tumor-targeting trans-splicingribozyme for cancer treatmentrdquo in Methods in Molecular Biol-ogy vol 1103 pp 83ndash95 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Recommended