YY1 Complex Promotes Quaking Expression via Super …Here we report that tran-scription of the QKI gene was activated by the Yin-Yang 1 (YY1)/p65/p300 complex, in which YY1 bound to

Molecular Cell Biology

YY1 Complex Promotes Quaking Expression viaSuper-Enhancer Binding during EMT ofHepatocellular CarcinomaJingxia Han1,2,3, JingMeng1,2,3, Shuang Chen2,3, XiaoruiWang4, Shan Yin5,Qiang Zhang2,3,Huijuan Liu2,3,4, Rong Qin1,3, Zhongwei Li1,3,Weilong Zhong1, Chao Zhang1, Heng Zhang1,3,Yuanhao Tang1,Tingting Lin6,Wanfeng Gao1, Xiaoyun Zhang1, Lan Yang2,3,Yanrong Liu2,3,Hong-gang Zhou1, Tao Sun1,2,3, and Cheng Yang1,3

Abstract

Quaking (QKI) is an alternative splicing factor that canregulate circRNA formation in the progression of epithelial–mesenchymal transition, but themechanism remains unclear.High expression of QKI is correlated with short survival time,metastasis, and high clinical stage and pathology grade inhepatocellular carcinoma (HCC). Here we report that tran-scription of the QKI gene was activated by the Yin-Yang 1(YY1)/p65/p300 complex, in which YY1 bound to the super-enhancer and promoter of QKI, p65 combined with thepromoter, and p300 served as a mediator to maintain thestability of the complex. This YY1/p65/p300 complexincreased QKI expression to promote the malignancy of HCC

as well as an increased circRNA formation in vitro and in vivo.Hyperoside is one of several plant-derived flavonol glycosidecompounds. Through virtual screening and antitumor activityanalysis, we found that hyperoside inhibited QKI expressionby targeting the YY1/p65/p300 complex. Overall, our studysuggests that the regulatorymechanismofQKI depends on theYY1/p65/p300 complex and that it may serve as a potentialtarget for treatment of HCC.

Significance: These findings identify the YY1/p65/p300complex as a regulator of QKI expression, identifying severalpotential therapeutic targets for the treatment of HCC.

IntroductionHepatocellular carcinoma (HCC) is one of the most preva-

lent and malignant tumors with a high mortality rate world-wide (1, 2). Metastasis is the most important cause of mortalityin HCC (3, 4). Epithelial–mesenchymal transition (EMT) playsa critical role in tumor progression (5, 6), and its pathologicactivation during tumor development can lead to the metastasisof primary tumors (3, 7, 8). CircRNAs are purposefully syn-thesized and implicated in specific biological roles in EMT.Quaking (QKI) is a distinct functional protein that mediatealternative splicing, which belongs to the STAR family of the

KH domain, containing RNA binding proteins. QKI regulate awide range of genes via alternative splicing and circRNA for-mation during EMT (9). Thus, it displays a potential role intumorigenesis (10). QKI is also associated with the develop-ment and progression of human cancer (11, 12). However, itspotential role in HCC and its regulatory mechanism in EMThave yet to be described.

Yin-Yang 1 (YY1) is a transcription factor involved in cancerprogression (13). YY1 can act as a transcriptional activator or arepressor in the regulation of gene expression (14, 15). Extensiveevidence indicates that YY1 is inversely correlatedwith E-cadherinexpression and crucial in EMT and tumor cell metastasis (16, 17).YY1 is also carcinogenic in various cancer types, such as breast andprostate cancer (18). YY1 is significantly upregulated in HCCtissues (1).

In this study, YY1 is highly expressed and positively correlatedwith QKI in patients with HCC with a short survival time. YY1binds to the super-enhancer and the promoter of QKI, p65combines with the promoter, and p300 serves as a mediator,causing the formation of DNA loops and leading to the abnormalactivation ofQKI. Abnormally activatedQKI causes the formationof circular RNA and the occurrence of EMT and tumor metastasisin HCC.

Materials and MethodsCell culture

All the cells were bought from the ATCC and KeyGen Biotech,cultured inDMEMor 1640medium supplementedwith 10%FBSand 1% penicillin–streptomycin solution at 37�C in 5% CO2

1State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy,Nankai University, Tianjin, China. 2Tianjin Key Laboratory for Evaluation ofPharmaceutical Property, Tianjin International Joint Academy of Biomedicine,Tianjin, China. 3Tianjin Key Laboratory of Molecular Drug Research, TianjinInternational Joint Academy of Biomedicine, Tianjin, China. 4College of LifeScience, Nankai University, Tianjin, China. 5OBiO Technology (Shanghai) Corp.,Ltd., China. 6Tianjin Medical University Eye Hospital, School of Optometry andOphthalmology, TMU, Tianjin Medical University Eye Institute, Tianjin, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J. Han, J. Meng, S. Chen, X. Wang, and S. Yin contributed equally to this article.

Corresponding Authors: Tao Sun, Nankai University, No. 38 Tongyan Road,Haihe River Education Park, Jinnan District, Tianjin 300350, China. Phone: 8613-5129-22691; Fax: 226-537-8009; E-mail: [email protected]; andCheng Yang, Nankai University, No. 38 Tongyan Road, Haihe River EducationPark, Jinnan District, Tianjin 300350, China. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-2238

�2019 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1451

on February 27, 2021. © 2019 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2238

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-18-2238&domain=pdf&date_stamp=2019-3-19

http://cancerres.aacrjournals.org/

atmosphere. The HCC cell lines were used within less a year andtested forMycoplasma before thawing original stocks. All the cellswere identified and periodically authenticated by morphologicinspection and biomarkers detection of hepatocellular carcino-ma, growth curve analysis.

Plasmid construction and transfectionpcDNA3.1–3 � Flag-YY1, pcDNA3.1–3 � Flag-p65,

pDONR223-EP300, pLKD-U6-shRNA, and pCD2.1-ciR plasmidswere obtained from Youbio, Obio Technology, and Geneseed.The QKI sequence was constructed into the pGL3 luciferasevectors (Promega) containing the luciferase gene under the con-trol of the SV40 promoter. siRNA was obtained from SANTAGenepharma. All of the plasmids and control vectors were trans-fected into the cells by using Lipofectamine 2000 (Invitrogen). Allof the constructs were prepared through PCR by using the appro-priate primers (Supplementary Table S1). The primers of theoverexpression vector are provided in Supplementary Table S2.

qRT-PCRTotal RNAwas extracted using Trizol reagent (Invitrogen) from

cells and tumor tissues. cDNAsynthesiswas performedwithOligo(dT) or random primers by using a Quantscript RT Kit (Tiangen).A SYBR RT–PCR kit (Tiangen) was used for transcript quantifica-tion with specific primers. Expression levels were quantified usingthe 2�DDCt methodwith b-actin as an internal control. The primersare provided in Supplementary Table S3.

Dual-luciferase reporter gene assayA pGL3 promoter vector containing different fragments of YY1

or p65 binding sites and YY1 or p65-overexpressed or QKIknockdown vectors were cotransfected into the HCC cells. After48 hours of transfection, luciferase activities were detected using aDual-Luciferase Reporter GeneAssay Kit (Promega) in accordancewith the manufacturer's instructions and normalized with Renillaluciferase activity. All of the experiments were performed intriplicates.

Immunopurification and silver stainingLysates from PLC-PRF-5 cells expressing Flag-YY1 were pre-

pared using 0.3% NP-40 lysis buffer [0.2 mmol/L EDTA,50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.3%Nonidet P-40] containing the protease inhibitor cocktail (Roche).Anti- Flag Tag (L5) Affinity beads (BioLegend) were incubatedwith the cell extracts for 12 hours at 4�C. After binding wascompleted, the beads were washed with cold 0.1% NP-40 lysisbuffer [0.2 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 7.4),150 mmol/L NaCl, and 0.1%NP-40]. Afterward, the Flag peptide(Sigma) was applied to the beads to elute the Flag proteincomplex. The eluents were collected and visualized through10% SDS-PAGE. Subsequently, silver staining was performedusing a Fast Silver Stain Kit (Beyotime). Distinct protein bandswere retrieved and analyzed through LC/MS-MS.

Immunoprecipitation and Western blot analysisIn immunoprecipitation, 50 mL of 50% protein A/G agarose

(Pierce) was incubated with control or specific antibodies at 4�Cwith constant rotation for 8 hours. PLC-PRF-5 cell lysates wereprepared by incubating the cells in 0.3% NP-40 lysis buffer in thepresence of protease inhibitor cocktails. Lysates were centrifugedat 12,000 rpm for 10minutes at 4�Cand incubatedwith antibody-

conjugated beads for additional 12 hours. After incubation wasperformed, the beads were washed five to six times by using cold0.1% NP-40 lysis buffer. The precipitated proteins were elutedfrom the beads by resuspending the beads in 2 � SDS-PAGEloading buffer and boiling for 10 minutes at 99�C. The boiledimmune complexes were subjected to SDS-PAGE and subsequentimmunoblotting. Antibody against p65 (1:100; Cell SignalingTechnology), YY1 (1:100; Cell Signaling Technology), and p300(1:100; Cell Signaling Technology).

The antibodies for Western blot analysis: QKI (1:1,000; Affin-ity), p300 (1:1,000; Affinity), E-cadherin (1:1,000; Affinity),vimentin (1:1,000; Affinity), and GAPDH (1:5,000; Affinity).

Fast protein liquid chromatography chromatographyPLC-PRF-5 cell extracts were applied to a Superdex 200 10/300

GL (GE Healthcare) equilibrated with 1� PBS. The column waseluted at a flow rate of 0.5 mL/minute, and fractions werecollected.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) experiments were

performed on 1 � 107 PLC-PRF-5 cells. The cells with differenttreatments were cross-linked using 1% formaldehyde (Sigma) for10 minutes, quenched with 0.25 mol/L glycine, and washed withcold PBS. The cell pellet was resuspended in 1 mL of cell lysisbuffer containing 1� Protease Inhibitor Cocktail II, incubated inice for 15minutes, dissociated by pipetting, and pelleted throughcentrifugation at 800 � g for 5 minutes at 4�C. The pellet wasresuspended in 1 mL of nuclear lysis buffer. Effective sonicationwas confirmed through bioanalyzer analysis. The chromatinfraction was incubated with an anti-YY1 monoclonal antibody(1:100; Abcam) at 4�C overnight. Protein/DNA complexes werereversed cross-linked to obtain free DNA. DNA was extracted andused for PCRamplificationwithQKI-specificprimers. Theprimersare provided in Supplementary Table S3.

Cell invasion assaysA cell invasion assay was performed using a 24-well Transwell

chamber (BD Biosciences). At 48-hour posttransfection, PLC-PFR-5 and cells were trypsinized and transferred to the Matrigel(BD Biosciences)-coated top chamber in 100 of serum-free medi-um. FBS was added to the bottom chamber as a chemoattractant.After 24 hours, the invasive cells on the bottom surface of thechamber were stained with 0.1% crystal violet and then counted.

Wound-healing assayAt 24-hour posttransfection, a straight scratchwas created in the

center of eachwell by using amicropipette tip. Cell migrationwasassessed bymeasuring the cellmovementwithin the scratch in thewell. The wound closure speed after 24 and 48 hours was deter-mined and normalized to length at 0 hour. Each experiment wasperformed in triplicate.

ImmunofluorescencePLC-PRF-5 cells were fixed with 4% paraformaldehyde for 20

minutes at room temperature and blocked with 5% BSA for 30minutes. The cells were incubated at room temperature withvimentin (1:200; Affinity) and E-cadherin antibodies (1:200;Affinity) for 1 hour and then with FITC- and TRITC-labeledsecondary antibodies (1:200; Earthox LLC) at room temperaturefor 1 hour. For each step, the cells were washed twice with PBS for

Han et al.

Cancer Res; 79(7) April 1, 2019 Cancer Research1452




5 minutes. The prepared specimens were counterstained withDAPI (Southern Biotechnology Associates) for 2 minutes andobserved under a confocal microscope (Nikon).

Scanning electron microscopyPLC-PRF-5 cells were fixed, dehydrated in acetone/isoamyl

acetate (1:1), and dried with a gradient concentration of aceto-nitrile. Gold-coated cells were photographed through scanningelectron microscopy (SEM; JEOL 6000).

Differential expression profilingPLC-PRF-5 cells were treated with YY1 or QKI overexpression

lysed with TRIzol Reagent (Invitrogen). Microarray was per-formed on the basis of a high-throughput gene expression profile.

Proximity ligation assaysProximity ligation assays (PLA) was conducted to detect and

quantify the amount of YY1–p65 protein complexes. The cellswere fixed for 15 minutes with 200 mL of 4% PFA. Permeabiliza-tion was carried out for 10 minutes with 200 mL of 0.1% TritonX-100. The coverslips were blocked and incubated with 30 to40 mL of diluted primary antibodies for 90 minutes. YY1–p65antibody pairs were used with a PLA Staining Development Kit(Sigma) in accordance with the manufacturer's instructions.Bound antibodies were detected using TRITC-labeled probesand fluorescent microscope.

Xenograft tumor modelEach BALB/c nude mouse in the control group was subcuta-

neously injected with approximately 1 � 106 PLC-PRF-5 andHep3B cells. The other groups were injected with cells with stablyexpressed p65 or YY1 to the posterior flank. Each group (Ctrl, p65,YY1, p65þYY1, QKI, and p65þYY1þsiQKI) consisted of fivemice. Tumor-bearing mice were treated when they reached anaverage tumor size of 100 to 200 mm3. The p65þYY1þsiQKIgroup was treated every 3 days with the intratumor injection ofshQKI viral suspension (50 mL) and then continuously injectedthrice. Tumor diameters were serially measured using a digitalcaliper every 3 days. Tumor volumes were calculated using thefollowing equation: length�width2/2. On day 24, the mice weresacrificed. Tumor tissues were collected, fixed with 10% formalin,and embedded in paraffin. The remaining tissues were kept in adeep freezer at �80�C for protein and RNA isolation. All of theanimal experiments were performed in accordance with theapproved protocols of the Institutional Animal Use and CareCommittee.

Experimental metastasis assayThe cells (2�105)were intravenously injected in the tail vein of

BALB/c nude mice. Each group (Ctrl, p65, YY1, p65þYY1, QKI,and p65þYY1þsiQKI) consisted of five mice. TheYY1þp65þsiQKI group was treated every 3 days with the intra-tumor injection of shQKI viral suspension (50 mL) and contin-uously injected thrice. After 8 weeks, the mice were sacrificed. Thelungs were collected for metastasis analysis.

Orthotopic (intrahepatic) injection of tumor cellsHep3B-Luc cells (1 � 106 cells/50 mL) were interhepatically

injected into BALB/c nude mice. The group information andsubsequent processing were the same as those in experimentalmetastasis assay. After 8 weeks, tumor burden was analyzed

through bioluminescence. The mice were intraperitoneallyinjected with 150 mg/kg of luciferin (Caliper Life Sciences) andimaged in accordance with the manufacturer's recommendationswith tumors facing the camera by using an IVIS200 (Caliper LifeSciences).

IHC assay and analysisThe mouse tissues were incubated with xylene for deparaffi-

nization and ethanol with decreased concentrations for rehy-dration. Afterward, 3% hydrogen peroxide was applied to blockthe endogenous peroxidase activity. The microwave antigenretrieval technique was utilized for antigen retrieval. Afterblocking was performed, the samples were incubated withthe following primary antibodies at 4�C overnight: QKI(1:200; Abcam), YY1 (1:200; Abcam), p65 (1:200; Affinity),E-cadherin (1:200; Affinity), and vimentin (1:200; Affinity).PBS displaced the primary antibody in the negative group.The secondary antibody was subsequently added using anHRP-polymer antimouse/rabbit IHC Kit (Maixin Biotech) atroom temperature for 1 hour. The samples were developedwith diaminobenzidine reagent, counterstained with hemat-oxylin, and mounted with permount33. The IHC score wascalculated by multiplying the intensity (0 ¼ negative, 1 ¼canary yellow, 2 ¼ claybank, and 3 ¼ brown) and the positivecell percentage scores (1 ¼ less than 25%, 2 ¼ 25%–50%, 3 ¼51%–75%, and 4 ¼ more than 75%).

Statistical analysisStatistical analyses were performed using GraphPad Prism

7 and SPSS v. 19. Statistically significant differences were calcu-lated by Student t test, one-way ANOVA, Mann–Whitney U test,Pearson's correlation, and Kaplan–Meier. P < 0.05was consideredsignificant.

ResultsYY1 complex plays a key role in QKI expression in HCC cells

PLC-PRF-5 cells were subjected to ChIP analysis to explore thetranscriptional regulation of YY1. The results revealed the pres-ence of binding sites on the upstream of QKI transcriptionalstarting site (TSS). Enriched YY1 motifs are displayed in Fig. 1A(up). ChIP-PCR analysis was carried out on PLC-PRF-5 cells byusing specific antibodies against YY1, showing the occupancy ofYY1 on the QKI promoter, which validated the ChIP-seq results(Fig. 1A, down). To assess the activity of YY1onQKI transcription,we performed a dual-luciferase reporter assay by transfectingPGL3-promoter plasmid that contained the binding motif of YY1alone or together with pcDNA3.1–3 � Flag-YY1 plasmid intoHCC cells. The results indicated that YY1 promoted the transcrip-tional activity of QKI (Fig. 1B).

We performed pull-down experiments to determine the keyinteraction of YY1 in vivo. The experiment showed that YY1 wasco-purified with a list of proteins, including p65 and p300(Fig. 1C). A Venn diagram was generated to identify the sharedproteins interacting with YY1, determine whether YY1 combinedwith other proteins to form a transcriptional complex, and con-sequently regulate the expression of QKI. A total of 82 proteinswere found in the intersection of the protein interacting with YY1by the FpClass database and proteins copurified with YY1, whichcontained p300. YY1 was included in predicted transcriptionfactors databases, which regulated QKI. Notably, the common

YY1 Activates QKI Expression by Super-Enhancer in HCC

www.aacrjournals.org Cancer Res; 79(7) April 1, 2019 1453




genes from the three protein types included p65 (Fig. 1D). Theinteraction network of 87 red-marked proteins of Fig. 1D wasshown (Fig. 1E). In addition, the presence of p65 and p300 in theYY1-associated protein complex was confirmed through Western

blot analysis on the column eluates (Fig. 1F). The results revealedthat interactions occurred among YY1, p65, and p300.

To further confirm the in vivo interactions among YY1, p65, andp300, we extracted total proteins from PLC-PRF-5 cells and

Figure 1.

YY1 is a key transcription factor for QKI expression in PLC-PRF-5 cells. A, ChIP-seq analysis on the DNA-binding motifs of YY1 on the promoters of QKI (top). ChIPexperiments on the QKI promoter by using antibodies against YY1 in PLC-PRF-5 cells. B,Dual-luciferase reporter assay in four HCC cells. The promoter activitiesof QKI were significantly increased because of overexpressed YY1. C, Pull-down analysis on YY1-associated proteins. Cellular extracts from Flag-YY1–expressingPLC-PRF-5 cells were subjected to affinity purification with anti-Flag affinity columns and eluted with Flag peptide. Elutes were resolved through SDS-PAGE andsilver stained.D, Venn diagram of transcription factors on the upstream of QKI prediction by using Chipbase database, proteins interacting with YY1 by usingFPclass database, and proteins interacting with YY1 by performing pulldown analysis. E, Protein–protein interaction network of 87 red-marked proteins in theintersection of D. F, Immunoblot for YY1, p65, and p300 by using pulldown products. G, Coimmunoprecipitation of endogenous YY1, p65, and p300 wasperformed in PLC-PRF-5 cells. H, Fast protein liquid chromatography experiments in PLC-PRF-5 cells. Chromatographic elution profiles and immunoblot analysisof the chromatographic fractions with antibodies against the indicated proteins are shown. Equal volumes from each fraction were analyzed, and the elutionpositions of the calibration proteins with knownmolecular masses (kDa) are indicated. I, Coexpression analyses on YY1, p65, and QKI in TCGA Database. Data arepresented as means� SD. �� , P < 0.01.

Han et al.





performed coimmunoprecipitation with antibodies exploring theendogenous proteins (Fig. 1G). The results demonstrated that YY1was efficiently coimmunoprecipitated with p65 and p300. Fastprotein liquid chromatography experiments were subsequently

performed using nuclear extracts. The elution patterns of p65 andp300 substantially overlapped with that of YY1 (Fig. 1H). Furtherstatistical analysis onTCGAdata showed that the expression levelsof YY1, p65, andQKI inHCC tumor tissueswere positively related

Figure 2.

YY1 enhances QKI gene transcription and translation through the formation of a complex with p65 and p300. A, Human QKI genomic locus with ChIP-Seq datafrom the Cistrome Data Browser database. B, Reporter assay constructs; summary of luciferase promoter and enhancer assays of YY1-binding sites (YBSs-Proand YBSs-SE) in PLC-PRF-5 cells. C,QKI promoter deletion cloned upstream of the luciferase gene in the pGL3 luciferase reporter. Activity values werenormalized to Renilla luciferase activity.D, Luciferase activities with JQ1 treatment. E,Western blot analysis detecting QKI expression with JQ1 treatment.F, Schematic of the mechanism through which YY1/p65/p300 regulates QKI.G,QKI expression measured throughWestern blot analysis in PLC-PRF-5 cellstransfected with YY1 or p65 alone or cotransfected with QKI siRNA. H,QKI expression measured by usingWestern blot analysis with the transfection ofYY1 or p65 siRNA.






between YY1 and QKI (r¼ 0.6239, P < 0.0001) and between p65and QKI (r ¼ 0.3118, P < 0.0001; Fig. 1I).

YY1 enhances QKI gene transcription and translationdepending on the formation of a complex with p65 and p300

Genome-wide studies have established that enhancers can bedefined as DNA sequences that bind to H3K4me1 and H3K27ac,and they are located on the upstream of known TSSs (19–21).Promoters can be defined as DNA sequences that bind toH3K4me3 and POLR2A. Fig. 2A shows the human QKI genomiclocus with ChIP-Seq data from the Cistrome Data Browser data-base for POLR2A, H3K4me3, H3K27ac, H3K4me1, YY1, p300,and p65. SE plays a critical role in gene expression (22). Therefore,we determined whether the expression of QKI is regulated by SE.QKI SE is located at 163,826,086 to 163,833,600 on chromosome6 (http://sea.edbc.org/). YY1 binds within this region. We foundthat YY1 bound to the SE and the promoter ofQKI, p65 bound tothe promoter, and p300weakly bound to the promoter ofQKI. Toconfirm the role of YY1 on QKI transcription, we cloned YY1-binding sites on the promoter (YBSs-pro) and YY1-binding siteson SE (YBSs-SE) into the pGL3 luciferase reporter. The resultsfurther confirmed that YY1 could bind to the SE and the promoterofQKI to improve the transcriptional activity ofQKI (Fig. 2B). Toidentify the binding sites for YY1, p65, and p300, we cloned 12deletion fragments of QKI into the pGL3 luciferase reporter.Notably, regions 5 and 8 exhibited the highest enhancer activity,and YY1 was overexpressed relative to that of the empty pGL3luciferase reporter. Region 12 presented the highest enhanceractivity with p65 overexpression. Conversely, p300 did not dis-play a remarkable increase in enhancer activity (Fig. 2C). p300, atranscriptional coactivator that interacts with various transcrip-tion factors, is a coactivator of YY1 and p65 in QKI gene expres-sion. SE is also implicated in gene expression (22). Therefore, wedetermined whether QKI expression is regulated by SE. We used asmallmolecule named JQ1,which can specifically disrupt SE (23).Luciferase assay showed that JQ1 could significantly reduce thepGL3 luciferase reporter luciferase activity that contained YY1motif (Fig. 2D). Western blot analysis results demonstrated thatJQ1 could reduce the QKI expression (Fig. 2E). Accordingly, theschematic showed the mechanism of QKI regulation by YY1through the formation of a complex with p65 and p300(Fig. 2F). Afterward, the effects of YY1 and p65 onQKI expressionwere verified.Western blot analysis results indicated that YY1 andp65 promoted the QKI expression level, and the QKI knockdownin YY1 or p65-overexpressing PLC-PRF-5 cells offset the promot-ing effect of YY1 and p65 onQKI expression (Fig. 2G). The silenceof YY1 and p65 repressed the QKI expression (Fig. 2H). Theseresults showed that YY1-p65-p300 promoted QKI transcriptionalactivity and gene expression by binding to QKI SE and promoter.

YY1 and p65 promote EMT, migration, and invasion bytargeting QKI

Western blot assay was conducted to explore whether YY1 andp65 regulate the EMTprocess by targetingQKI. The results showedthat YY1 and p65 improved the expression of vimentin andreduced the expression of E-cadherin. QKI knockdown in YY1and p65-overexpressing PLC-PRF-5 cells offset the effects of YY1and p65 on vimentin and E-cadherin expression (Fig. 3A). Theeffects of YY1 and p65 on cell phenotypes were observed throughSEM. Cell pseudopodia were increased, cell morphology wasconverted from an epithelial phenotype to a mesenchymal phe-

notype in YY1 or p65-overexpressing cells, and epithelioid wasrecovered after QKI depleted (Fig. 3B). Immunofluorescenceanalysis revealed that the E-cadherin expression significantlydecreased, whereas the vimentin expression increased when YY1,p65, or QKI was overexpressed. The QKI knockdown in YY1 andp65-overexpressing PLC-PRF-5 cells blocked the influence ofYY1 and p65 on E-cadherin and vimentin expression (Fig. 3C).Similarly, the QKI overexpression could also downregulateE-cadherin expression and upregulate vimentin expression.

These results indicated that YY1 and p65 promoted EMT inHCC cells. Therefore, they might also intensify the promotingeffects of QKI on migration and invasion. We selected two HCCcell lines, PLC-PRF-5 andHep3B, to verify our hypothesis throughwound-healing and invasion assays. In PLC-PRF-5 and Hep3Bcells, migration (Fig. 3D) and invasion (Fig. 3E) abilities signif-icantly increased in response to YY1 or p65-overexpressing treat-ment. QKI overexpression alone had similar effects. In QKI-siRNA-transfected cells, even with YY1 or p65 treatment, migra-tion and invasion were maintained at basal levels, which weresimilar to that in nontreated cells. These results collectivelyimplied that knockdownQKI could affect the YY1 or p65-inducedphenotype of HCC metastasis.

To assess the influence of YY1 on key biological functions, weanalyzed the whole transcriptome in YY1-treated cells. We con-ducted gene set enrichment analysis (GSEA) by using differen-tially regulated genes. Differentially expressed genes were sub-jected to Kyoto Encyclopedia of Genes andGenomes (KEGG) andGO analyses. After YY1 transfection was performed, VEGF, TGFb,Wnt, proliferation, and biological processes related to stem celldifferentiation were upregulated. In contrast, p53, apoptosis, andcell death were downregulated (Fig. 3F and G). These resultssupported the theory that YY1 complex effectively enhanced EMTin HCC cells by promoting the QKI expression.

QKI is related to EMTprogression and promotes the generationof circRNAs

To assess the effects of QKI on key biological functions, weconducted a whole-genome expression spectrum analysis. TotalRNAwas extracted from the PLC-PRF-5 cells transfectedwithQKI,and the whole genome expression chip was analyzed. To deter-mine the biological functions and pathways affected by differen-tially regulated genes, we subjected data to GO analysis. Figure 4Ashows that cell proliferation, migration, angiogenesis, cellularresponse to VEGF stimulus, and NIK/NF-kB-related biologicalprocesses were upregulated after QKI transfection. On the con-trary, apoptosis and adhesion biological processes were down-regulated. The upregulated genes associated with proliferation-,migration-, and angiogenesis-related biological processes afterQKI treatment are presented in Fig. 4B.

To examine the relationship among circRNA expression andHCC, YY1, or QKI expression, we searched the whole genomeexpression spectrumdata and compared the difference in circRNAexpression between each group through cluster analysis (Fig. 4C–E).We analyzed the common circRNAswith expression levels thatwere largely affected relatively by upregulation with YY1 or QKIoverexpression.

We also analyzed the common circRNAs with expression levelshighly affected by upregulation with YY1 or QKI overexpression(Fig. 4F). circRNAs exhibited a significant overlap in the twogroups. To examine the high-order properties of inter-ceRNAsignaling, we identified the optimal ceRNA pairs and constructed

Han et al.




http://sea.edbc.org/


the optimal ceRNA regulatory network (Fig. 4G). Our resultsshowed that YY1 increased the QKI expression to promote abun-dant circRNA formation. YY1 participated in promoting theoccurrence and development in HCC by targeting QKI throughthe proposed ceRNA mechanism. We constructed a circRNA-

miRNA-target gene network to visualize their interactions. cir-cRNAs adsorbed miRNAs and promoted oncogene expression. Inthe network, 14 miRNAs ranked relatively high, and 162 of themost likely target genes of these miRNAs were collected. Amongthese miRNAs, miR-6860 and miR-296 were the top predicted

Figure 3.

YY1 and p65 promote EMT, migration, and invasion by targeting QKI.A, Expression of E-cadherin and vimentin transfected with YY1, p65, and QKI orcotransfected with QKI siRNA. B,Morphological changes in PLC-PRF-5 cells transfected with YY1, p65, and QKI or cotransfected with QKI siRNA observed withSEM. C, Representative immunofluorescence images of E-cadherin and vimentin of PLC-PRF-5 cells treated in the samemanner as in the wound-healing assay. D,Migration of PLC-PRF-5 cells transfected with YY1, p65, and QKI or cotransfected with QKI siRNA. E, Cell invasion assays performed in PLC-PRF-5 cellstransfected with YY1, p65, and QKI or cotransfected with QKI siRNA. F, GSEA and KEGG analyses of the enriched expression of YY1 target gene sets after YY1overexpression in PLC-PRF-5 cells (red, YY1; blue, control). G, GSEA and GO analyses on the enriched expression of YY1 target gene sets after YY1 overexpression(red, YY1; blue, control). Data are presented as means� SD. � , P < 0.05; �� , P < 0.01.






miRNA targets of mRNA. The expression of MAPK signaling-pathway-related genes, namely, CACNB3, AKT2, ARRB1,CACNA1H, ELK4, FGF11,DUSP3, CACNB1, and FGF22, increased.

We screened six circRNAs that were upregulated evidently withYY1 and QKI overexpression. We conducted qRT-PCR to furtherverify the influences of YY1 and QKI on circRNA expression(Fig. 4H). The results demonstrated that six circRNAs improvedat different levels in response to YY1 overexpression. In QKIknockdown-treated cells, circRNA expression was maintained atbasal levels even with YY1 overexpression. YY1 indirectly con-trolled the expression of circRNAs by regulating the expressionof QKI. We also detected that QKI caused the formation ofcirc-0008150, which adsorbs miRNAs (miR-615-5p) targeted

vimentin to decrease vimentin expression (Supplementary Fig.S1A–S1C). circ-0007821, which can absorb miR-381-3p targetedZeb1(Supplementary Fig. S2A–S2C), was also increased underQKIoverexpressed cell. The whole-genome expression spectrum analy-sis showed thatEMT-related transcriptional factors, especiallyZeb1,was increased in QKI overexpressed cells (Supplementary Fig. S3).The results showed that QKI promotes vimentin expression byupregulated circ-0008150 absorbing miR-615-5p. Meanwhile,QKI indirectly inhibits E-cadherin expression by upregulatedZeb1, which regulated by circ-0007821 absorbing miR-381-3p.The effects of circ0008150, circ0007821, and circ0029308 on cellinvasion were also assessed. The results indicated that circ0008150and circ0007821 increased cell invasion ability (Fig. 4I).

Figure 4.

QKI is related to EMT progression, and it promotes the generation of circRNAs. A, Signaling pathways promoted and suppressed by QKI in HCC cells. B,Upregulated genes associated with proliferation, migration, and angiogenesis in PLC-PRF-5 cells after QKI treatment analyzed by using DAVID. C–E, Heatmap ofidentified circRNAs with changes between groups. F, Venn diagram of the upregulated circRNAs with YY1 or QKI overexpression.G, Graphical view of thecircRNA–miRNA–mRNA network of the upregulated circRNAs after QKI and YY1 overexpression. Purple circles, red circles, blue circles, and edges correspond tocircRNAs, miRNAs, mRNAs, and direct interaction links, respectively. H, qRT-PCR of six circRNAs performed in PLC-PRF-5 cells. I, Cell invasion assays performedin PLC-PRF-5 cells transfected with circRNAs.

Han et al.





QKI expression is required for the YY1 complex to promote themetastasis and malignancy of HCC

To gain further insights into the effects of the YY1 complextargeting QKI on tumor growth, EMT, and metastasis, we usedsubcutaneous PLC-PRF-5 and Hep3B tumor models. The resultsshowed that p65, YY1, or QKI increased tumor volume andknockdown QKI under p65 and YY1 overexpressed cells inhibitsthe promotion effect on tumor growth (Fig. 5A). The number ofmetastatic lung nodules increased in overexpression of YY1, p65,or QKI groups. QKI interference maintained the number ofmetastatic lung nodules at the same level as that of the controlgroup (Fig. 5B). We measured the expression levels of QKI, YY1,p65, E-cadherin, and vimentin of the tumors through IHC. HighYY1 and p65 expression levels significantly increased the expressionof QKI and vimentin but inhibited the expression of E-cadherin.QKI knockdown counteracted the effects of YY1 and p65 onE-cadherin and vimentin expression but did not affect YY1 andp65 expression (Fig. 5C and D). QKI expression was positivelycorrelated with YY1 and p65 in tumor tissues (Fig. 5E). Further-more, the circ0008150 and circ0007821 expression in each groupwas detected through qRT-PCR (Fig. 5F). High expression levels ofYY1 and p65 resulted in increased expression levels of circRNAs.

To examine whether QKI contributed to YY1 complex-promoted metastasis in vivo, we conducted experimental meta-stasis model by intravenous injection and divided the samegroups as previously described. The result showed that the averagenumber of metastatic lung nodules was significantly increasedin the highly expressed YY1 or p65 groups compared with thecontrol orQKI knockdown group (Fig. 5G andH). In intrahepaticinjection model, every BALB/c mouse were intrahepaticallyimplanted with 1 � 106 Hep3B-Luc cells. After 8 weeks, tumorburden was analyzed through bioluminescence imaging. Theresult showed that YY1, p65, and QKI could promote the intra-hepatic metastasis of HCC. The knockdown of QKI on the baseof YY1 and p65 overexpression maintained the metastasis atthe same level as that of the control group (Fig. 5I). These findingsconfirmed that YY1, p65, andQKI could expedite the intrahepaticmetastasis.

QKI is upregulated by YY1 complex, and it can promote themalignant progression of HCC

Clinical data analysis was carried out to further explore the roleof YY1 complex and its target gene QKI in HCC. Cancer tran-scription analysis on TCGA samples from UALCAN database(http://ualcan.path.uab.edu/index.html) showed that YY1 washighly expressed in HCC compared with that in normal livertissues (Supplementary Fig. S4A). Survival analysis on the expres-sion of YY1 showed that the high level of YY1 (P < 0.0001) wasassociated with poor overall survival in patients with HCC (Sup-plementary Fig. S4B). YY1 expression was positively correlatedwith the clinical stage and pathologic grade (Supplementary Fig.S4C and S4D). Further statistical analysis showed that patientswith high expression of p65 and YY1 in TCGAdatabase hadworseprognosis thanother patients (Fig. 6A). These datawere consistentwith the roles of YY1 and p65 in accelerating HCC development.The representative IHC images of normal liver tissues andHCC forQKI expression levels are shown in Fig. 6B. QKI was highlyexpressed in HCC compared with that in normal liver tissues(Fig. 6C). The QKI expression was positively correlated withclinical stage and pathologic grade (Fig. 6D and E). TheKaplan–Meier survival curve revealed that the increased QKI

expression indicated poor survival in patients with HCC(Fig. 6F). High YY1 and QKI expression levels were associatedwith short overall survival. This result was also observed in p65and QKI (Fig. 6G). These data were consistent with the role of theYY1 complex in promoting the QKI expression to facilitate HCCprogression.

Hyperoside inhibits EMT and metastasis of HCC by targetingYY1 complex

The detailed binding mode of the YY1 complex is shownin Fig. 7A. YY1 and p300 had a hydrogen bond interaction(Asp1625-Arg314), p65 and p300 had one hydrogen bond inter-action (Ser1232-Asn1196), YY1 and p65 had three hydrogenbond interactions (Gly96-Thr348, Ser61-Arg371, and Ser39-Thr356). According to the structures of YY1, p65, and p300, leadcompounds were selected from the Traditional Chinese Medicinedatabase through virtual screening. Hyperoside obtained themost excellent docking score with the YY1 complex (Fig. 7B).Hyperoside is one of the flavonol glycoside compounds fromnatural plant. The binding mode of hyperoside inhibited the YY1complex (Fig. 7C). To determine the effect of hyperoside on theYY1 complex, we performed coimmunoprecipitation with anantibody against YY1, followed by IB with anti-YY1, p65, andp300 (Fig. 7D). The results demonstrated that the interactionbetween YY1 and p65 or p300 was weakened after 50 mmol/Lhyperoside treatment. In addition, in situproximity ligation assays(PLA) was performed by using YY1 and p65 primary antibodiesand species-specific PLA probes. The results also revealed that YY1and p65 interactions were reduced under hyperoside treatment;however, QKI could not restore the reduced interaction betweenYY1 and p65 under hyperoside treatment (Fig. 7E). Western blotanalysis further demonstrated that hyperoside could decrease theQKI expression to inhibit EMT and did not affect YY1 and p65expression. Although, the overexpression of QKI could mitigatethe effects of hyperoside (Fig. 7F). We also investigated theinfluence of hyperoside on migration and invasion. The hypero-side treatment of the PLC-PRF-5 cells decreased cellmigration andinvasion, but the overexpression of QKI could counteract thisinhibitory effect (Fig. 7G and H). qRT-PCR results showed thathyperoside suppressed the level of circ-0008150 and circ-0007821, and QKI restored the expression to normal levels(Fig. 7I). Collectively, these experiments supported the notionthat hyperoside suppressed QKI and circRNAs expression toinhibit EMT.

The nude mice that were subcutaneously injected withPLC-PRF-5 cells underwent hyperoside treatment to verify theeffect of hyperoside against HCC.Hyperoside treatment inhibitedthe tumor growth in a dose-dependent manner, and the over-expression ofQKI couldmitigate the effects of hyperoside (Fig. 7J).The survival curve revealed that the survival times of hyperosidetreatment groups were longer than those of the control group(Fig. 7K). Metastatic lung nodules also decreased after hypero-side treatment (Fig. 7L). The QKI overexpression could coun-teract the therapeutic effect of hyperoside. We further measuredthe QKI, E-cadherin, and vimentin expression levels in tumortissues through IHC (Fig. 7M). The results showed that hypero-side treatment was associated with increased the E-cadherinexpression and decreased the QKI and vimentin expressionlevels compared with those of the control group. In thehyperosideþQKI group, E-cadherin and vimentin expressionwere similar to that of the control group. The measurement





http://ualcan.path.uab.edu/index.html


Figure 5.

QKI expression is required for the YY1 complex to promote the metastasis and malignancy of HCC. A, Comparison of PLC-PRF-5 and Hep3B tumor growth in micewere measured starting 6 days after implantation. All of the groups were euthanized on day 24. B,Metastatic lung nodules were counted in each group. C,QKI,p65, YY1, E-cadherin, and vimentin expression levels in the tumor tissues of p65, YY1, p65þYY1, QKI, and p65þYY1þsiQKI groups analyzed through IHC staining.D, IHC staining index of QKI, YY1, p65, E-cadherin, and vimentin in HCC tissues. E, Correlation analysis between p65 and QKI (P¼ 0.0012) and YY1 and QKI (P¼0.0048). F, qRT-PCR analysis of circ-0008150 and circ-0007821 levels in tumor tissues. G, Representative figures of hematoxylin and eosin staining on the serialsections of metastatic tumors in the lungs. H,Metastatic lung nodules were counted in each group. I, Representative images of in vitro BLI. Quantification of thesignal intensity in each group. Quantified values are shown in total flux (photons/second). Data are presented as means� SD. � , P < 0.05; �� , P < 0.01.

Han et al.





of the circRNA expression in tumors as determined throughqRT-PCR indicated that hyperoside efficiently inhibitedcirc-0008150 and circ-0007821 expression. circRNA expressionreturned to normal in the hypþQKI group (Fig. 7N). Overall,these experiments indicated that hyperoside could block EMTand metastasis by targeting the YY1 complex at the interfaceof the YY1 complex, and the overexpression of QKI couldmitigate the effects of hyperoside.

DiscussionYY1 is functionally diverse because it is a transcription pro-

moter and transcriptional repressor. YY1 also participates in EMTand contributes to cancer progression (13, 15, 24, 25). YY1stimulates the expression of the transcription factor Snail (26),which in turn causes cells to undergo EMT by directly repressingmetastasis-suppressor gene products, such as E-cadherin (27) andclaudins (28), and inducing metastasis-inducer gene products,

such as vimentin (29). High YY1 expression suggests the wors-ening condition of malignant tumors and poor prognosis. Inpancreatic ductal adenocarcinoma, YY1 suppresses cell invasionand metastasis by downregulating MMP10 (30). In melanoma,YY1 contributes to cell proliferation, cell-cycle progression,migra-tion, and cell invasion (31).

Here, we found that YY1 is highly expressed in HCC cells,thereby playing a crucial role in EMT program. YY1 overexpres-sion in HCC may be involved in the regulation of progressionfrom cirrhosis to HCC and malignant progression (32). In ourresults, YY1 expression is also positively correlated with theclinical stage and pathological grade of patients with HCC. Thesefindings indicated that YY1 served as a functional biomarker inHCC progression. YY1 can form transcriptional complexes withtranscription factors and a homologous dimer to exert transcrip-tional activation effects (33). YY1 dimer is combined with thepromoter and enhancer to form an enhancer–promoter loop,causing an abnormal gene transcription (34). YY1 combinedwith

Figure 6.

QKI is upregulated by YY1 complex, and it can promote the malignant progression of HCC. A, Survival analysis of TCGA samples for the relationship betweensurvival time of HCC patients and p65 and YY1 expression levels. B, Representative QKI expression images of normal liver tissues and HCC samples. C,QKIexpression level in HCC tumors and normal liver tissues based on TCGA dataset. D, Analysis on the QKI expression levels in TCGA LIHC samples based on anindividual clinical stage. E, Analysis on the QKI expression levels in TCGA HCC samples based on an individual pathologic grade. F, Kaplan–Meier plots of theoverall survival rate of patients with HCCwith QKI low or high expression levels (P¼ 0.0001).G, Kaplan–Meier plots of the overall survival rate of patients withHCC with YY1/QKI low or high levels (left), and with p65/QKI low or high levels (right) (P < 0.0001). Data are presented as means� SD. �� , P < 0.01.






Figure 7.

Hyperoside inhibits the EMT andmetastasis of HCC by targeting YY1 complex.A, Detailed binding mode of YY1, p65, and p300. B, Prediction score of dockingbetween small-molecule drugs and YY1 complex. C, Left, the structure of hyperoside. Right, predicted binding modes of hyperoside (green) and YY1/p65/p300complex.D, Coimmunoprecipitation of endogenous YY1, p65, and p300was performed in PLC-PRF-5 cells treated with 120 mmol/L hyperoside. E, The effect ofhyperoside on YY1 and p65 interaction assessed by using Duolink proximity assay. F,Western blot analysis with antibody against p65, YY1, QKI, E-cadherin, andvimentin under different treatments. Western blots were also probed for GAPDH as a loading control. G–H, Migration and invasion of PLC-PRF-5 cells withdifferent treatments. I, qRT-PCR analysis on circ-0008150 and circ-0007821 levels under hyperoside and QKI treatment. J, Representative images and tumorgrowth curves of nudemice in four groups. K, Survival curve of nude mice in four groups. L,Metastatic lung nodules were counted in each group.M, IHC stainingindex of QKI, E-cadherin, and vimentin in HCC tissues in different groups.N, Expression of circ-0008150 and circ-0007821 analyzed by using qRT-PCR. Data arepresented as means� SD. � , P < 0.05; �� , P < 0.01.

Han et al.





p300 triggers histone acetylation, which causes gene activation byfacilitating the binding of RNA polymerase (35). YY1 can formtranscriptional complexeswith transcription factors and a homol-ogous dimer to exert a transcriptional activation effect (34). Ourresults showed that YY1 combined with the super-enhancer andthe promoter ofQKI in the YY1-p65-p300 complex to trigger EMT.Themechanism is different from that of the homologous dimer ofYY1, but the function is consistent. In this YY1-p65-p300,whetherYY1 binds to two DNA sites in the form of a monomer or ahomologous dimer still needs further verification. In our study,transcriptome analysis showed that VEGF-, TGFb-, and Wnt-related signaling pathways were upregulated with QKI overex-pression. By contrast, p53- and apoptosis-related signaling path-ways were downregulated.

QKI is associated with adult cancers potentially through itsregulation of microRNA functionality (36). For example, QKIfunctions as a tumor suppressor in colon cancer (37). In ourstudy, high QKI expression was correlated with poor prognosisin HCC. YY1 promoted the expression of QKI by forming atranscriptional complex with p65 and p300. YY1 acceleratedthe expression of circRNAs by targeting QKI and caused abun-dant circRNA formation. These processes promoted tumorprogression in part by its ability to induce EMT (38–40). QKIaffects pre-mRNA splicing, mRNA turnover, and translation inmany cancers. QKI is sufficient and necessary to mediateplasticity between epithelial and mesenchymal states and driveEMT-associated alternative splicing by promoting exon skip,even in the absence of EMT inducers (41). Simon J. Connshowed that QKI dynamically regulates abundant circRNAformation by the alternative splicing factor during EMT. Theabundance of circRNA is dependent on intronic QKI bindingmotifs. CircRNAs are purposefully synthesized and implicatedin specific biological roles in EMT (9). QKI may regulate a widerange of genes via alternative splicing and circRNA formationduring EMT. In our study, differential expression profilinganalysis demonstrated that QKI caused the formation ofEMT-related circRNAs, such as circ-0008150 and circ-0007821.Circ-0008150, which adsorbed miR-615-5p and targetedvimentin, thereby decreasing its expression level. CircRNA-0007821, which could absorb miR-381-3p targeted Zeb1, alsoincreased in QKI-overexpressing cells. Consequently, Zeb1increased to inhibit E-cadherin expression. The overexpressionof QKI also promoted the expression of other EMT-relatedtranscriptional factors, such as Twist and Snail families, whichdecreased E-cadherin by combining E-box and promotingvimentin expression. YY1 silencing downregulated QKI andcircRNA expression. Many studies have shown that the expres-sion profiles of miRNAs and circRNAs are abnormal in manycancer types, and many of them have focused on their epige-netic regulation in cancer development (2, 42). The abnormalexpression of circRNAs is commonly observed in various cancertypes (21). Certain circRNAs present EMT-related functions;thus, they may affect mesenchymal cell properties, such asmigration, invasion, and propensity for cancer to metastasis.QKI regulated hundreds of alternative splicing targets and the

formation of circRNAs to exert pleiotropic effects, such asincreasing cell migration and invasion and promoting EMT.Hyperoside, one of the flavonol glycoside compounds fromnatural plant, specifically inhibits the formation of theYY1/p65/p300 complex by targeting the complex interactioninterface and subsequently depresses the transcription of QKIand the formation of downstream circRNA. Therefore, hypero-side may inhibit HCC EMT.

In conclusion, our studies displayed the regulatory mechanismof YY1 in the QKI expression in HCC. Specifically, a novelmechanism presented that the YY1/p65/p300 complex regulatedthe QKI expression by binding to the SE and the promoter topromote abundant circRNAs formation and promote EMT inHCC. Thus, YY1 was a potential biomarker of HCC, therebyimproving the accelerated effects of QKI in HCC tumorigenesisin vitro and in vivo. YY1 could also be used as an independentpredictor of metastasis and survival in HCC. In brief, the eluci-dation of the roles and regulatory mechanisms of the YY1/p65/p300 complex might facilitate the treatments for the suppressionof cancer cell metastasis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Meng, S. Chen, T. Sun, C. YangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Han, X. Wang, Q. Zhang, H. Liu, R. Qin,C. Zhang, L. YangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Han, J. Meng, Z. Li, W. Zhong, H. Zhang, Y. Tang,W. Gao, X. Zhang, Y. Liu, H.-g. Zhou, T. SunWriting, review, and/or revision of the manuscript: J. Han, J. Meng, S. Chen,T. Sun, C. YangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Yin, H. Liu, T. Lin, T. SunStudy supervision: T. Sun, C. Yang

AcknowledgmentsThis work was supported by National Natural Science Funds of China (grant

nos. 81572838, 81872374, 81402973, 81703581, 81871972), Tianjin Scienceand Technology Project (grant nos. 15PTGCCX00140, 18PTSYJC00060),Chinese National Major Scientific and Technological Special Project for"Significant New Drugs Development" (grant nos. 2018ZX09736-005,SQ2018ZX090201), The National Key Research and Development Program ofChina (grant no. 2018YFA0507203), Postdoctoral support scheme for inno-vative talents (grant no. BX20180150), China Postdoctoral Science Foundation(grant no. 2018M640228), The Fundamental Research Funds for the CentralUniversities, Nankai University, and Natural Science Foundation for YoungScholar of Tianjin (grant no. 16JCQNJC12300).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 20, 2018; revised December 13, 2018; accepted February 6,2019; published first February 13, 2019.

References1. Dong S, Ma X, Wang Z, Han B, Zou H, Wu Z, et al. YY1 promotes HDAC1

expression and decreases sensitivity of hepatocellular carcinoma cells toHDAC inhibitor. Oncotarget 2017;8:40583–93.

2. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, et al.MicroRNA expression profiles classify human cancers. Nature 2005;435:834–8.






3. Moreno-Bueno G, Portillo F, Cano A. Transcriptional regulation of cellpolarity in EMT and cancer. Oncogene 2008;27:6958–69.

4. Tang ZY, Ye SL, Liu YK, Qin LX, SunHC, Ye QH, et al. A decade's studies onmetastasis of hepatocellular carcinoma. J Cancer Res ClinOncol 2004;130:187–96.

5. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancerstatistics, 2012. CA Cancer J Clin 2015;65:87–108.

6. Lee GA, Hwang KA, Choi KC. Roles of dietary phytoestrogens on theregulation of epithelial-mesenchymal transition in diverse cancer metas-tasis. Toxins 2016;8. doi: 10.3390/toxins8060162.

7. GuptaGP,Massague J. Cancermetastasis: building a framework. Cell 2006;127:679–95.

8. Thiery JP. Epithelial-mesenchymal transitions in development and pathol-ogies. Curr Opin Cell Biol 2003;15:740–6.

9. Conn SJ, Pillman KA, Toubia J, Conn VM, Salmanidis M, Phillips CA, et al.The RNA binding protein quaking regulates formation of circRNAs. Cell2015;160:1125–34.

10. Hall MP, Nagel RJ, FaggWS, Shiue L, Cline MS, Perriman RJ, et al. Quakingand PTB control overlapping splicing regulatory networks during musclecell differentiation. RNA 2013;19:627–38.

11. Xi Z, Wang P, Xue Y, Shang C, Liu X, Ma J, et al. Overexpression of miR-29areduces the oncogenic properties of glioblastoma stem cells by down-regulating Quaking gene isoform 6. Oncotarget 2017;8:24949–63.

12. de Miguel FJ, Pajares MJ, Martinez-Terroba E, Ajona D, Morales X, SharmaRD, et al. A large-scale analysis of alternative splicing reveals a key role ofQKI in lung cancer. Mol Oncol 2016;10:1437–49.

13. Wang J, Zhou L, Li Z, Zhang T, LiuW, Liu Z, et al. YY1 suppresses FEN1over-expression and drug resistance in breast cancer. BMC Cancer 2015;15:50.

14. Bonavida B, Kaufhold S. Prognostic significance of YY1 protein expressionand mRNA levels by bioinformatics analysis in human cancers: a thera-peutic target. Pharmacol Therapeut 2015;150:149–68.

15. Shi JM, Hao AX, Zhang Q, Sui GC. The role of YY1 in oncogenesis and itspotential as a drug target in cancer therapies. Curr Cancer Drug Tar 2015;15:145–57.

16. Baritaki S, Rapozzi V, Pelayo R, VegaM,Huerta-Yepez S, Bonavida B. Yin Yang1 (YY1) regulates EMT via transcriptional activation of Snail and its over-expression in cancer stem cells (CSCs) in prostate cancer: clinical significance.Mol Cancer Ther 2013;12. doi: 10.1158/1535-7163.TARG-13-A53.

17. Bonavida B, Baritaki S, Huerta-Yepez S, Vega MI, Chatterjee D, Yeung K.Novel therapeutic applications of nitric oxide donors in cancer: roles inchemo- and immunosensitization to apoptosis and inhibition of metas-tases. Nitric Oxide 2008;19:152–7.

18. Wang WX, Yue ZN, Tian ZP, Xie YR, Zhang JM, She YP, et al. Expression ofYin Yang 1 in cervical cancer and its correlation with E-cadherin expressionand HPV16 E6. PLoS One 2018;13:e0193340. doi: 10.1371/journal.pone.0193340.

19. Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, et al.Distinct and predictive chromatin signatures of transcriptional promotersand enhancers in the human genome. Nat Genet 2007;39:311–8.

20. ViselA, BlowMJ, Li Z, Zhang T, Akiyama JA,Holt A, et al. ChIP-seq accuratelypredicts tissue-specific activity of enhancers. Nature 2009;457:854–8.

21. Zhang X, Zhu M, Yang R, Zhao W, Hu X, Gan J. Identification andcomparison of novel circular RNAs with associated co-expression andcompeting endogenous RNA networks in pulmonary tuberculosis.Oncotarget 2017;8:113571–82.

22. Ke L, Zhou H, Wang C, Xiong G, Xiang Y, Ling Y, et al. Nasopharyngealcarcinoma super-enhancer-driven ETV6 correlates with prognosis. PNAS2017;114:9683–8.

23. Yang R, Wu Y, Ming Y, Xu Y, Wang S, Shen J, et al. A super-enhancermaintains homeostatic expression of Regnase-1. Gene 2018;669:35–41.

24. Wang CC, Chen JJ, Yang PC. Multifunctional transcription factor YY1: atherapeutic target in human cancer? Expert Opin Ther Targets 2006;10:253–66.

25. Kim JS, Son SH, Kim MY, Choi D, Jang IS, Paik SS, et al. Diagnostic andprognostic relevance of CP2c and YY1 expression in hepatocellular carci-noma. Oncotarget 2017;8:24389–400.

26. PalmerMB,Majumder P, Cooper JC, YoonH,Wade PA, Boss JM. Yin yang1regulates the expression of snail through a distal enhancer. Mol Cancer Res2009;7:221–9.

27. Nieto MA. The snail superfamily of zinc-finger transcription factors.Nat Rev Mol Cell Biol 2002;3:155–66.

28. Liu B, Dong H, Lin X, Yang X, Yue X, Yang J, et al. RND3 promotes Snail 1proteindegradation and inhibits glioblastoma cellmigration and invasion.Oncotarget 2016;7:82411–23.

29. Myong NH. Loss of E-cadherin and acquisition of vimentin in epithelial-mesenchymal transition are noble indicators of uterine cervix cancerprogression. Korean J Pathol 2012;46:341–8.

30. Zhang JJ, ZhuY, Xie KL, PengYP, Tao JQ, Tang J, et al. Yin Yang-1 suppressesinvasion and metastasis of pancreatic ductal adenocarcinoma by down-regulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mecha-nism. Mol Cancer 2014;13:130.

31. Zhao G, Li Q, Wang A, Jiao J. YY1 regulates melanoma tumorigenesisthrough a miR-9 � RYBP axis. J Exp Clin Cancer Res 2015;34:66.

32. Gordon SA,Huerta-Yepez S, VegaM, Bonavida B. Nuclear expression of thetranscription factor yin yang 1 in hepatocellular carcinoma: Potential forprognostic significance. Liver Transplant 2006;12:C49.

33. Gordon S, Akopyan G, Garban H, Bonavida B. Transcription factorYY1: structure, function, and therapeutic implications in cancer biology.Oncogene 2006;25:1125–42.

34. Weintraub AS, Li CH, Zamudio AV, Sigova AA, Hannett NM, Day DS, et al.YY1 is a structural regulator of enhancer-promoter loops. Cell 2017;171:1573–88.

35. Lee JS, Galvin KM, See RH, Eckner R, Livingston D, Moran E, et al. Relief ofYY1 transcriptional repression by adenovirus E1A is mediated by E1A-associated protein p300. Genes Dev 1995;9:1188–98.

36. Jain P, Resnick AC. MYB-QKI drives childhood brain tumors via tripartitemechanism. Cell Cycle 2017;16:390–1.

37. YangG, FuH, Zhang J, Lu X, Yu F, Jin L, et al. RNA-binding protein quaking,a critical regulator of colon epithelial differentiation and a suppressor ofcolon cancer. Gastroenterology 2010;138:231–40.

38. Yuan JH, Yang F, Wang F, Ma JZ, Guo YJ, Tao QF, et al. A long noncodingRNA activated by TGF-beta promotes the invasion-metastasis cascade inhepatocellular carcinoma. Cancer Cell 2014;25:666–81.

39. Li XM, Tang ZY, Zhou G, Lui YK, Ye SL. Significance of vascular endothelialgrowth factor mRNA expression in invasion and metastasis of hepatocel-lular carcinoma. J Exp Clin Cancer Res 1998;17:13–7.

40. Yu H, Shen H, Zhang Y, Zhong F, Liu Y, Qin L, et al. CAV1 promotes HCCcell progression and metastasis through Wnt/beta-catenin pathway.PLoS One 2014;9:e106451.

41. Pillman KA, Phillips CA, Roslan S, Toubia J, Dredge BK, Bert AG,et al. miR-200/375 control epithelial plasticity-associated alternativesplicing by repressing the RNA-binding protein Quaking. EMBO J2018;37. doi: 10.15252/embj.201899016.

42. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, et al. Amammalian microRNA expression atlas based on small RNA librarysequencing. Cell 2007;129:1401–14.


Han et al.




2019;79:1451-1464. Published OnlineFirst February 13, 2019.Cancer Res Jingxia Han, Jing Meng, Shuang Chen, et al. Binding during EMT of Hepatocellular CarcinomaYY1 Complex Promotes Quaking Expression via Super-Enhancer

Updated version

10.1158/0008-5472.CAN-18-2238doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2019/02/13/0008-5472.CAN-18-2238.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/79/7/1451.full#ref-list-1

This article cites 39 articles, 4 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/79/7/1451.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/79/7/1451To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-18-2238

http://cancerres.aacrjournals.org/content/suppl/2019/02/13/0008-5472.CAN-18-2238.DC1

http://cancerres.aacrjournals.org/content/79/7/1451.full#ref-list-1

http://cancerres.aacrjournals.org/content/79/7/1451.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/79/7/1451


Documents

YY1 Complex Promotes Quaking Expression via Super …Here we report that tran-scription of the QKI gene was activated by the Yin-Yang 1 (YY1)/p65/p300 complex, in which YY1 bound to