Long Noncoding RNA PVT1 Promotes Non Small Cell Lung … · NOP2 nucleolar protein (NOP2; refs.18, 19). In our previous study, we also found that increased PVT1 expression promoted

Cancer Biology and Signal Transduction

Long Noncoding RNA PVT1 Promotes Non–SmallCell Lung Cancer Cell Proliferation throughEpigenetically Regulating LATS2 ExpressionLi Wan1,2, Ming Sun3, Guo-Jian Liu1, Chen-Chen Wei1, Er-Bao Zhang3, Rong Kong3,Tong-Peng Xu4, Ming-De Huang2, and Zhao-Xia Wang1

Abstract

Long noncoding RNAs (lncRNA) are a novel class of tran-scripts with no protein coding capacity, but with diverse func-tions in cancer cell proliferation, apoptosis, and metastasis. ThelncRNA PVT1 is 1,716 nt in length and located in the chr8q24.21region, which also contains the myelocytomatosis (MYC) onco-gene. Previous studies demonstrated that MYC promotes PVT1expression in primary human cancers. However, the expressionpattern and potential biologic function of PVT1 in non–smallcell lung cancer (NSCLC) is still unclear. Here, we found thatPVT1 was upregulated in 105 human NSCLC tissues comparedwith normal samples. High expression of PVT1 was associatedwith a higher tumor–node–metastasis stage and tumor size, as

well as poorer overall survival. Functional analysis revealed thatknockdown of PVT1 inhibited NSCLC cell proliferation andinduced apoptosis both in vitro and in vivo. RNA immunopre-cipitation and chromatin immunoprecipitation assays demon-strated that PVT1 recruits EZH2 to the large tumor suppressorkinase 2 (LATS2) promoter and represses LATS2 transcription.Furthermore, ectopic expression of LATS2 increased apoptosisand repressed lung adenocarcinoma cell proliferation by regu-lating the Mdm2-p53 pathway. Taken together, our findingsindicated that PVT1/EZH2/LATS2 interactions might serve asnew target for lung adenocarcinoma diagnosis and therapy.Mol Cancer Ther; 15(5); 1082–94. �2016 AACR.

IntroductionLung cancer is a predominant cause of cancer-related death

worldwide, due to its high morbidity and the lack of effectivetherapy strategies (1). Non–small cell lung cancer (NSCLC)accounts for approximately 85% of all lung cancer cases (2).Despite recent advancements in clinical and experimental oncol-ogy, the prognosis of NSCLC remains poor, with a 5-year overallsurvival rate of around 11% (3). Because NSCLC is a multigenedisorder, the biologic processes involved inNSCLC tumorigenesisare complicated. Thus, a detailed understanding of the mechan-isms and molecular pathways underlying NSCLC developmentandprogression is essential to improve diagnosis, prevention, andtreatment of NSCLC patients.

The rapid development in sequencing techniques and improve-ment in bioinformatics has led to the discovery of Long noncodingRNA (lncRNA), which are more than 200 bp in length and widelytranscribed across the eukaryotic genome (4, 5). LncRNAs wereonce regarded as transcriptional "noise" or cloning artifacts becauseof their absence of protein-encoding ability (6). Emerging evidencehas indicated that aberrant expression of lncRNAs is associatedwith numerous diseases, including diabetes (7, 8). Importantly,lncRNAs were shown to act as regulators of tumorigenesis viaparticipating in biologic processes, such as modulation of apopto-sis, proliferation, drug resistance, and the epithelial–mesenchymaltransition process (9–12). For example, our previous study dem-onstrated that the lncRNAHox transcript antisense intergenic RNA(HOTAIR) contributes to cisplatin resistance of human lung ade-nocarcinoma cells via downregulation of p21WAF1/CIP1 expression.However, the contributions of aberrant lncRNAs to NSCLC devel-opment and progression and the underlying molecular pathwaysor mechanisms are not well documented.

Chromatin-modifying complexes play important roles inlncRNA-mediated epigenetic regulation of gene expression.Recent studies showed that 24% of lncRNAs, one category oflncRNAs, are expressed in various cell types and bound by poly-comb repressive complex 2 (PRC2), which comprises EZH2,SUZ12, and EED and acts as a molecular scaffold for histonemodification complexes (13, 14). These lncRNAs recruit PRC2 tothe promoter of target genes and catalyze the trimethylation oflysine residue 27 of histone 3 (H3K27me3). For example, RASSF1antisense 1 (ANRASSF1) recruits PRC2 to the RASSF1A promoter,resulting in silenced expression of RASSF1A and increased cellproliferation.Ubiquitin-conjugating enzymeE2K (UBE2K) acts asa negative prognostic factor for lymph node metastasis andsurvival via associating with PRC2 in bladder cancer (15, 16).

1Department ofOncology, SecondAffiliatedHospital, NanjingMedicalUniversity, Nanjing, People's Republic of China. 2Department ofOncology, Huai'an First People's Hospital, Nanjing Medical University,Huai'an, People's Republic ofChina. 3Department ofBiochemistry andMolecular Biology, Nanjing Medical University, Nanjing, People'sRepublic of China. 4Department of Oncology, First Affiliated Hospital,Nanjing Medical University, Nanjing, People's Republic of China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

L. Wan, M. Sun, and G.-J. Liu contributed equally to this article and should beregarded as joint first authors.

Corresponding Author: Zhao-Xia Wang, Second Affiliated Hospital, NanjingMedical University, 121 Jiangjiayuan Road, Nanjing 210011, People's Republic ofChina. Phone: 025-58509810; Fax: 25-58509994; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0707

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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These data suggest that the dysregulation of PRC2-relatedlncRNAs is involved in carcinogenesis.

PVT1, an lncRNA that is 1,716 nt in length, is located in thechr8q24.21 region which also contains the myelocytomatosis(MYC) oncogene. Interestingly, MYC protein leads to the accu-mulation of PVT1 in primary human cancers (17). In addition,high expression of PVT1 is a prognostic indicator for colorectalcancer patients and suppresses colorectal cancer cell apoptoticability.Moreover, PVT1 could promote the proliferation and stemcell–like property of hepatocellular carcinoma cells by stabilizingNOP2 nucleolar protein (NOP2; refs.18, 19). In our previousstudy, we also found that increased PVT1 expression promotedcell proliferation through binding with EZH2 and repressing P15and P16 transcription in gastric cancer (20). Although Yang andcolleagues have demonstrated that PVT1 may serve as an "onco-gene" in NSCLC cells (21), but the potential molecular mechan-isms and downstream pathways of PVT1 in NSCLC cells are stillunknown. In this study, we investigated PVT1 expression inNSCLC tissues and cell lines, and explored its biologic effectsand mechanisms in NSCLC cells in vitro and in vivo.

Materials and MethodsTissue specimens and clinical data collection

We obtained 105 paired NSCLC and adjacent nontumor lungtissues from patients who underwent surgery at The First andSecond Affiliated Hospital of Nanjing Medical University from2010 to 2011. The patients were diagnosed with NSCLC (stages I,II, and III) based on histopathologic evaluation. The clinicopath-ologic characteristics of the NSCLC patients are summarizedin Table 1. The patients did not receive any local or systemictreatment before operation. All collected tissue samples wereimmediately snap-frozen in liquid nitrogen and stored at �80�Cuntil RNA extraction. The ethics committee of The Second Affil-iated Hospital of Nanjing Medical University approved the studyprotocol.

Cell linesWe purchased six NSCLC cell lines (PC-9, SPC-A1, H157,

H1299, A549, and SK-MES-1) and the normal bronchial epi-thelial cell line 16HBE from the Institute of Biochemistry andCell Biology of the Chinese Academy of Sciences (Shanghai,China). A549 and H1299 cells were cultured in RPMI-1640,and 16HBE, PC-9, SPC-A1, H157, and SK-MES-1 cells werecultured in DMEM medium (GIBCO-BRL) supplemented with10% FBS (Gibco),100 U/mL penicillin sodium, and 100 mg/mL streptomycin sulfate at 37�C in a humidified air atmospherecontaining 5% CO2. All cell lines were authenticated by shorttandem repeat DNA profiling.

RNA extraction and qPCR assaysTRIZOL reagent was used to extract total RNA from tissue

samples or cells according to the manufacturer's instructions.Total RNA (500 ng) was reverse transcribed in a final volume of10 mL using random primers under standard conditions with thePrimeScript RTReagentKit (TaKaRa).TomeasurePVT1 expressionlevels, we used SYBR Premix Ex Taq (TaKaRa) following themanufacturer's instructions. GAPDH expression was used fornormalization. Primer sequences are listed in SupplementaryTable S1. Real-time PCR was performed in triplicate on an ABI7500, and data were calculated using the comparative cyclethreshold (CT) (2�DDCT) method.

RNA interferenceA549 and PC-9 cells were transfected with siRNA using Lipo-

fectamine 2000 (Invitrogen), and the cells were incubated for 48hours before use in assays. The siRNA sequences are listed inSupplementary Table S1. The shRNA PVT1 (CCCAACAGGAG-GACAGCTT) was cloned into the pENTR/U6 vector.

Plasmid generationThe LATS2 sequence was synthesized and subcloned into the

pCDNA3.1 vector (GENECHEM) to generate the pCDNA–LATS2vector for ectopic expression in cells. pCDNA3.1 vector was usedas a control. The qPCR assaywas conducted to evaluate expressionof LATS2. We synthesized 1 to 400 bp, 1 to 800 bp, and 1 to 1,200bp regions of PVT1 and subcloned into the pCDN3.1 vector,respectively (Real Gene).

Cell viability assaysThe Cell Proliferation Reagent Kit I (MTT; Roche Applied

Science) was used to test the cell viability. A549 and PC-9 cellsplated in 96-well plates (3,000 cells/well) were transfectedwith si-PVT1. We estimated the cell viability every 24 hours according tothe manufacturer's protocol. The MTT experiments were con-ducted in quadruplicate. For colony formation assay, transfectedcells (n ¼ 1200) were placed in 6-well plates and maintained inproper medium containing 10% FBS for 2 weeks. The mediumwas replaced every 4 days, and visible colonies were counted after2 weeks.

Flow cytometric analysisWe harvested A549 and PC-9 cells that were transfected with

si-PVT1 for 48 hours. Double staining with FITC–Annexin Vand propidium iodide was performed using the FITC Annexin VApoptosis Detection Kit (BD Biosciences) according to themanufacturer's recommendations. Cells were discriminatedinto viable cells, dead cells, early apoptotic cells, and apoptotic

Table 1. Correlation between PVT1 expression and clinicopathologiccharacteristics of NSCLC patients (n ¼ 105)

CharacteristicsPVT1 Low number

of case (%)High numberof case (%)

x2 test(P value)

Age (years)>65 27 (55.1) 28 (50.0) 0.696�65 22 (44.9) 28 (50.0)

GenderMale 32 (65.3) 30 (53.6) 0.24Female 17 (34.7) 26 (46.4)

Histologic subtypeSquamous cell carcinoma 28 (57.1) 35 (62.5) 0.69Adenocarcinoma 21 (42.9) 21 (37.5)

TNM stageIa þ Ib 22 (44.9) 8 (14.3) 0.001a

IIa þ IIb 16 (32.7) 19 (33.9)IIIa 11 (22.4) 29 (51.8)

Tumor size�5 cm 32 (65.3) 20 (35.7) 0.003a

>5 cm 17 (34.7) 36 (64.3)Lymph node metastasisNegative 30 (61.2) 20 (35.7) 0.011a

Positive 19 (38.8) 36 (64.3)Smoking historySmokers 36 (73.5) 33 (58.9) 0.087Never smokers 13 (26.5) 23 (41.1)

aOverall P < 0.05.

PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

www.aacrjournals.org Mol Cancer Ther; 15(5) May 2016 1083




cells, and the relative ratio of early apoptotic cells was com-pared with control transfection from each experiment. For cell-cycle analysis, cells were stained with propidium oxide usingthe CycleTEST PLUS DNA Reagent Kit (BD Biosciences) fol-lowing the manufacturer's protocol and analyzed by FACScan.The percentages of cells in G0–G1, S, and G2–M phases werecounted and compared.

Western blot assay and antibodiesProtein lysates were separated by 15% SDS-PAGE and trans-

ferred to 0.22-mm NC membranes (Sigma) and incubated withspecific primary antibodies. P53 andMdm2 antibodies (1:1,000)were purchased from SAB, Inc. Anti-LATS2 (1:1,000) was pur-chased from Abcam. GAPDH antibody was used as a control.

lncRNA expression from GEO DataSetsNSCLCgene expressiondatawere obtained fromGEODataSets

(GDS). Four independent data sets from GSE18842 (22),GSE19188 (23), GSE19804 (24), and GSE30219 (25) wereincluded in this study. The raw CEL files were downloaded fromGEO database and normalized using Robust Multichip Average(RMA). After we downloaded probe sequences from GEO ormicroarray manufacturers, blastþ2.2.30 was used to reannotatesprobe on GENCODE Release 21 sequence databases for lncRNAandmRNA (26). For multiple probes corresponding to one gene,maximumnormalized signal was selected to generate expressionsof lncRNA and mRNA. Two-sample t test or paired-sample t testaccording to experimental design was employed as differentialexpression callingmethod, followed by the Benjamini–Hochberg(FDR) adjustment. For verifying expression correlation betweengenes, Pearson correlation analysis was used after CEL files fromGSE43580 (27) and GSE31210 (28) were downloaded andnormalized by RMA.

Tumor formation assay in a nude mouse modelMale athymic BALB/c mice (5-week-old) were maintained

under specific pathogen-free conditions andmanipulated accord-ing to protocols approved by the Shanghai Medical ExperimentalAnimal Care Commission. A549 cells stably transfected with sh-PVT1 or empty vector were harvested at a concentration of 2� 107

cells/mL, and 0.1 mL was subcutaneously injected into the flanksof the nude mice, at one injection per mouse. Tumor growth wasmonitored, and tumor sizes and weights were measured every 3days. Tumor volume was calculated using the formula, volume¼(length�width2� 0.5). At 21 days after injection, the mice werekilled and tumor weights were measured. The primary tumorswere excised, and tumor tissues were used for qRT-PCR analysis ofPVT1 expression levels and immunostaining analysis of Ki-67protein expression.

Subcellular fractionationThe separation of nuclear and cytosolic fractions was per-

formed using the PARIS Kit (Life Technologies) according to themanufacturer's instructions.

RNA immunoprecipitation assaysA549 and PC-9 cells were lysed for immunoprecipitation (IP)

of endogenous PRC2 complexes from whole-cell extracts. Theprotein A Sepharose beads were incubated with positive anti-body SNRNP70, negative antibody IgG, and interested antibodyEZH2 (Milipore) for 30 minutes at 4�C. Then, the supernatants

of whole-cell extracts were incubated with treated beads for6 hours at 4�C. We used the wash buffer to wash the beads for6 times. To isolate the RNA–protein complexes from beads, thebeads were incubated with 0.1% SDS/0.5 mg/mL Proteinase Kfor 30 minutes at 55�C. The qPCR assays were used to furtherevaluate the PRC2 isolated from the IP materials.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were con-

ducted using the EZ-CHIP KIT according to the manufacturer'sinstructions (Millipore). EZH2 antibody was obtained fromAbcam. H3 trimethyl Lys 27 antibody was obtained from Milli-pore. The ChIP primer sequences are listed in SupplementaryTable S1. Quantification of immunoprecipitated DNA was per-formed using qPCR with SYBR Green Mix (TaKaRa). EZH2 couldbind to the 366 region of LATS2 promoter. ChIP data werecalculated as a percentage relative to the input DNA.

RNA pulldown assaysThe pCDNA3.1-PVT1 vector was cut by restriction enzymes

Nru I and treated with RNase-free Dnase I (Biolabs). LncRNA-PVT1 was transcribed from cutted vector by mMESSAGEmMACHINE T7 Kit (Ambion) and purified with an RNeasyMini kit (Qiagen) in vitro. We biotin labeled the 30 end oflncRNA-PVT1 referencing to the instruction of Pierce RNA 3�End Desthiobiotinylation Kit (Thermo Scientific). One milli-gram of protein from A549 and PC-9 cell extracts were thenmixed with 50 pmol of biotinylated RNA, incubated with 50mL of magnetic beads for 1 hour at 4�C (Thermo Scientific).The RNA–protein complex was isolated from magnetic beadsby Biotin Elution Buffer and boiled in SDS buffer for 10minutes. The retrieved protein was detected using the standardWestern blot technique.

ImmunohistochemistryThe primary tumors were immunostained for Ki-67 as previ-

ously described (29).

Statistical analysisAll statistical analyses were performed using SPSS 20.0

software (IBM). The significance of differences betweengroups was estimated by the Student t test, Wilcoxon test,or c2 test. Disease-free survival and overall survival rates werecalculated by the Kaplan–Meier method with the log-rank testapplied for comparison. The date of survival was evaluated byunivariate and multivariate Cox proportional hazards mod-els. Variables with P < 0.05 in univariate analysis were used insubsequent multivariate analysis on the basis of Cox regres-sion analyses. Kendall Tau-b and Pearson correlation analyseswere conducted to investigate the correlation between PVT1and LATS2 expressions. Two-sided P values were calculated,and a probability level of 0.05 was chosen for statisticalsignificance.

ResultsPVT1 expression is upregulated and correlated with a poorprognosis of NSCLC

First, we analyzed the profiles of NSCLC patient from GeneExpression Omnibus (GEO) and found that PVT1 was upre-gulated in NSCLC tissues compared with normal lung tissues

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(Fig. 1A). To explore whether PVT1 was differently expressed inNSCLC tissues, a total of 105 paired NSCLC tissue samples andadjacent normal counterparts were evaluated for PVT1 expres-sion using qPCR assay. The results revealed that PVT1 expres-sion was upregulated in 75 tumor tissues (Fig. 1B). We used themedian expression of PVT1 as a cutoff point to divide allpatients into two groups: the high PVT1 group (n ¼ 56, foldchange �3.0) and the low PVT1 group (n ¼ 49, fold change�3.0). Statistical analysis revealed that PVT1 expression levelsin NSCLC were significantly correlated with tumor size (P ¼0.003), advanced pathologic stage (P ¼ 0.001), and lymphnode metastasis (P ¼ 0.011). However, PVT1 expression wasnot associated with other factors including sex (P ¼ 0.24) andage (P ¼ 0.696) in NSCLC (Table 1).

Association between PVT1 expression and patient survivalNext, we conducted a Kaplan–Meier survival analysis to explore

the correlation between PVT1 expression and NSCLC patientprognosis. Progression-free survival was 38.2% for the low PVT1group, and 17.0% for the high PVT1 group. Median survival timefor the lowPVT1groupwas 31months, and15months for the highPVT1 group (Fig. 1D). As shown in Fig. 1E, the overall survival rateover 3 years for the low PVT1 group was 44.5%, and 25.7% for thehighPVT1 group. Themedian survival time for the lowPVT1 groupwas 33 months, and 18 months for the high PVT1 group.

Univariate survival analysis showed that lymph node metas-tasis, tumor–node–metastasis (TNM) stage, and PVT1 expressionlevel could be viewed as prognostic factors (Table 2). Otherclinicopathologic features, including sex and age, were not

Figure 1.Relative PVT1 expression in NSCLC tissues and its clinical significance. A, relative PVT1 expression in the profiles of NSCLC patient tissue from GEO. B, relativeexpression of PVT1 in NSCLC tissues (n ¼ 105) and in paired adjacent normal tissues (n ¼ 105). PVT1 expression was examined by qPCR assays andnormalized to GAPDH expression. Results are presented as the fold change in tumor tissues relative to normal tissues. C, the patients were divided into two groupsaccording to the median value of relative PVT1 expression. D and E, Kaplan–Meier analysis of progression-free survival (PFS) or overall survival (OS)according to PVT1 expression levels.






statistically significant prognostic factors. Moreover, multivariateCox regression analyses showed that expression of PVT1(P ¼ 0.012), along with TNM stage (P ¼ 0.024), was an inde-pendent prognostic factor for NSCLC patients (Table 2).

Knockdown of PVT1 inhibits NSCLC cell proliferation, inducesapoptosis, and promotes cell-cycle arrest

Previous study showed that MYC protein could lead to theaccumulation of PVT1 in primary human cancers; hence, weanalyzed the relationship between PVT1 and MYC in lung cancer

cell lines (from The Cancer Genome Atlas data) and NSCLCtissues (GSE28571 and GSE43850). We found that the PVT1expression level is positively related with MYC expression bothin cells and tissues (Supplementary Fig. S1).

Wenext investigated thebiologic effect ofPVT1onNSCLCcells.First, a qPCR assay was performed to evaluate the expression ofPVT1 in various NSCLC cell lines. As shown in Fig. 2A, four celllines (A549, PC-9, SK-MES-1, and H157) expressed higher levelsof PVT1 compared with the normal bronchial epithelial cell line(16HBE). In contrast, the relative expression level of PVT1 was

Figure 2.Effects of knockdown of PVT1 onNSCLC cell viability in vitro. A, PVT1expression levels of NSCLC cell lines(A549, PC-9, SK-MES-1, H157, SPCA1,and H1299) compared with that innormal human bronchial epithelial cells(16HBE). B, A549 and PC-9 cells weretransfected with si-PVT1. C, MTT assayswere conducted to determine the cellproliferation ability for si-PVT1–transfectedA549 andPC-9 cells. Valuesindicate the mean � SD from threeindependent experiments. D, colony-forming assays were performed todetermine the proliferation ofsi-PVT1–transfected A549 and PC-9cells. � , P < 0.05; �� , P < 0.01; NS,not significant.

Table 2. Univariate and multivariate analysis of overall survival in NSCLC patients (n ¼ 105)

Univariate analysis Multivariate analysisVariables HR (95% CI) P value HR (95% CI) P value

Age 1.375 (0.753–2.510) 0.299Gender 1.238 (0.679–2.259)) 0.486Smoker 1.344 (0.736–2.453) 0.336Histologic subtype 0.938 (0.513–1.717) 0.837Chemotherapy 0.739 (0.425–1.287) 0.285Tumor size 1.742 (0.947–3.203) 0.074Lymph node metastasis (no vs. yes) 2.042 (1.105–3.776) 0.023a 1.429 (0.739–2.764) 0.288TNM stage (IIIa vs. I or II) 2.010 (1.373–2.941) <0.001a 1.629 (1.067–2.489) 0.024a

PVT1 expression (high vs. low) 3.151 (1.613–6.154) 0.001a 2.464 (1.214–4.999) 0.012a

Abbreviation: CI, confidence interval.aOverall P < 0.05.

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lower in H1299 cell lines. Next, we knocked down endogenousPVT1 expression in A549 and PC-9 cells by siRNAs. At 48 hoursafter transfection, PVT1 expression was reduced by approximately78.1% or 81.9% compared with control siRNA-transfected cells(Fig. 2B). MTT and colony formation assays demonstrated thatgrowth of A549 and PC-9 cells transfected with si-PVT1 wasattenuated compared with control cells (Fig. 2C and D). Further-more, flow cytometry analysis revealed that knockdown of PVT1expression induced apoptosis and cell-cycle arrest at G1–G0 phasein A549 and PC-9 cells (Fig. 3A–D).

Knockdown of PVT1 inhibits NSCLC cell tumorigenesisin vivo

To further investigate whether reduction of PVT1 expressioncould impact tumor growth in vivo, A549 cells stably transfectedwith sh-PVT1 or empty vectors were inoculated into male nudemice. Eighteen days after injection, the tumor size in the sh-PVT1 group was decreased substantially compared with thecontrol group (Fig. 4A and B). The mean tumor weight of sh-

PVT1was significantly lower than that in the control group (Fig.4C). Next, we used a qPCR assay to explore the averageexpression of PVT1 in tumor tissues (Fig. 4D). As shownin Fig. 4E, immunohistochemistry (IHC) analysis confirmedthat the tumors formed from A549/sh-PVT1 cells displayedlower Ki-67 staining than those formed from the control cells.Our results indicated that knockdown of PVT1 expression couldsuppress tumor growth in vivo.

LATS2 is a key downstream mediator of PVT1To explore the underlying target genes of PVT1 in NSCLC, we

analyzed previously published gene expression profile down-stream of PVT1 in colorectal cancer cells by Takahashi andcolleagues (18).The qPCR assay was performed to detect theexpression of the cyclin B1 (CCNB1), ankyrin repeat and GTPasedomainArfGTPase activating protein 11 (ACAP11), SMADfamilymember 4 (SMAD4), which were mentioned in the earlier study.Unexpectedly, the qPCR results showed that PVT1 knockdowndid not affect the expression of these genes in A549 andPC-9 cells,

Figure 3.The effect of PVT1 on NSCLC cell cycle and apoptosis in vitro.A and B, cell cycle of A549 and PC-9 cells was analyzed by flow cytometry. The bar chart represents thepercentage of cells in G0-G1, S, or G2–M phase, as indicated. C and D, A549 and PC-9 cells were stained and analyzed by flow cytometry. LR, early apoptoticcells; UR, terminal apoptotic cells. Error bars, mean � SEM. All experiments were conducted in biologic triplicates with three technical replicates. � , P < 0.05;�� , P < 0.01.






but increased the expression of LATS2 (Fig. 5A). To independentlyverify this result, we conducted Western blot analysis to measureLATS2 expression. The results revealed that LATS2 protein levelswere also increased in si-PVT1–transfected cells (Fig. 5B).

PVT1 inhibits LATS2 expression by binding to EZH2Next, we investigated the mechanism by which PVT1 regu-

lates LATS2 expression. First, qPCR assays were performed toevaluate PVT1 expression in nuclear and cytosolic fractionsfrom A549 and PC-9 cells. GAPDH and U1 RNA were used asfractionation indicators. The results demonstrated higherexpression of PVT1 in the nucleus versus the cytosol in bothcell lines (Fig. 5D).

PVT1 might play a major regulatory function at the tran-scriptional level. Approximately 24% of lncRNAs physicallyassociate with PRC2. Thus, we next evaluated whether PVT1could bind to the PRC2 complex. RNA–protein interactionprediction (RPISeq) analysis showed that the PVT1-EZH2 andSUZ12-SUZ12 interaction scores were 0.87 and 0.83 withSupport Vector Machine classifier, respectively. Predictionswith probabilities >0.5 were considered positive, and accuraciesof the prediction ranged from 57% to 99% in independentdatasets of RNA–protein interactions (30).

Next, RNA immunoprecipitation (RIP) experiments wereperformed in A549 and PC-9 cell extracts using antibodiesagainst EZH2 and SUZ12. We used qPCR assay to detectRNA levels in immunoprecipitates. As shown in Fig. 6A, theresults revealed PVT1 enrichment in EZH2-RNA precipitates,whereas less enrichment was observed in SUZ12-RNA pre-

cipitates. Furthermore, we conducted RNA pulldown assaysin A549 and PC-9 cells to determine whether EZH2 isassociated with PVT1. The results revealed that EZH2 couldinteract with PVT1 (Fig. 6B). Moreover, we synthesized threeregions of PVT1, including 1 to 400 bp, 1 to 800 bp, and 1to 1,200 bp. Then, RNA pulldown assays were performed toconfirm which region of PVT1 could bind to EZH2, and theresults showed that 1 to 400 bp of PVT1 is the majorbinding site (Fig. 6C).

LATS2 is a tumor suppressor gene that is mutated in NSCLC,and downregulation of LATS2 could promote NSCLC cellviability and motility (31, 32). A previous study showed thathypermethylation of the promoter regions of LATS2 played avital role in the downregulation of its mRNA levels in NSCLC(33). EZH2, a component of PRC2, could mediate H3K27me3modification and epigenetically silence target genes. To furtherdetermine whether EZH2 can directly bind the promoter regionof LATS2, we designed four pairs of primers across 1,000 bp ofthe promoter region. CHIP assays confirmed that EZH2 couldbind to the LATS2 promoter region (Fig. 6D). Moreover,knockdown of PVT1 reduced EZH2 binding to LATS2 promoterregions (Fig. 6E). Finally, we analyzed the correlation betweenPVT1 and LATS2 expressions in NSCLC patient profiles fromGEO and found that there was a significantly negative corre-lation (Fig. 6F).

LATS2 may be involved in the Mdm2-P53 pathwayTo further investigate whether LATS2 is involved in the PVT1-

induced increase of NSCLC cells apoptosis and cycle arrest, we

Figure 4.The effect of knockdown of PVT1 on tumorigenesis in vivo. A and B, empty vector or sh-PVT1 was transfected into A549 cells, which were injected in the malenude mice (n ¼ 6), respectively. Tumor volumes were calculated after injection every 3 days. Bars, SD. C, tumor weights are represented as mean oftumor weights � SD. D, qPCR assay was performed to determine the average expression of PVT1 in xenograft tumors (n ¼ 6). E, the tumor sections were underhematoxylin and eosin (H&E) staining and IHC staining using antibodies against Ki-67. ��, P < 0.01.

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performed gain-of-function assays. The qPCR assay results con-firmed that LATS2 expression was significantly upregulated inA549 cells transfected with pCDNA–LATS2 compared with thecontrol group (Fig. 7A). MTT and colony formation assays dem-onstrated that the NSCLC cell viability was inhibited dramaticallyupon overexpression of LATS2 (Fig. 7B and C). Moreover, flowcytometry analysis showed a G0-G1 cell-cycle arrest and increasedapoptosis rate in A549 cells transfected with pCDNA–LATS2(Fig. 7D and E). A previous report showed that LATS2 could bindMdm2 to inhibit its E3 ligase activity and activate p53 (34).Thus,we performed Western blot analysis of Mdm2 and p53 expres-sions, and the results showed that Mdm2 expression wasdecreased and p53 expression was increased in A549 cells trans-fected with pCDNA–LATS2. Cells with knocked down PVT1showed consistent results (Fig. 7F). Thus, these findings suggestthat PVT1 could participate in the Mdm2-p53 pathway by epi-genetically regulating LATS2.

Moreover, we conducted rescue assays to determine whetherPVT1 contributes to NSCLC cell proliferation and apoptosisvia inhibiting LATS2 expression. A549 cells were cotransfectedwith si-PVT1 and si-LATS2. As shown in Fig. 8A, si-LATS2transfection could partly rescue si-PVT1–increased LATS2expression (Fig. 8A). The results of MTT and colony formationassays indicated that the proliferation ability of A549 cellscotransfected with si-PVT1 and si-LATS2 was improved com-paring with A549 cells transfected with si-PVT1 (Fig. 8B and

C). Meanwhile, apoptosis rate and cycle arrest also wererescued in cotransfected group comparing with si-PVT1 group(Fig. 8D and E). These dates indicate that PVT1 exertingbiologic effect on NSCLC cells may partly through repressingLATS2 expression.

DiscussionRecently, a large number of studies have demonstrated that

lncRNA dysregulation is involved in epigenetics and participatesin carcinogenesis. For instance, lncRNAHOTAIR promotes gastriccancermetastasis through inhibition of Poly r(C) Binding Protein(PCBP) 1 (35). In our previous studies, we found that p53-regulated lncRNA TUG1 affected NSCLC cell proliferation viaepigenetically regulatingHOXB7 expression.We also showed thatEZH2-mediated epigenetic suppression of lncRNA SPRY4-IT1promoted NSCLC cell proliferation and metastasis by affectingthe epithelial–mesenchymal transition. However, as the lncRNAresearch field has expanded quickly andmore lncRNAs have beencharacterized, important roles for aberrant lncRNAs in the devel-opment and metastasis of multiple cancers have been demon-strated. Therefore, the contributions of misregulated lncRNAs toNSCLC, and their biologic function andunderlyingmechanism inNSCLC cells should be investigated.

In this study, we found that another lncRNA PVT1 is upregu-lated in NSCLC tissues, and increased PVT1 expression is

Figure 5.PVT1 could inhibit LATS2 expression. A, the qPCR assay was conducted to detect the levels of LATS2 mRNA in A549 and PC-9 cells transfectedwith si-PVT1, and results are expressed relative to the corresponding values for control cells. B, Western blot assay was conducted to detect the levelof LATS2 protein in A549 or PC-9 cells transfected with si-PVT1. C, Western blot and qPCR assays were used to detect the LATS2 expressionboth in mRNA and protein levels in A549 and PC-9 cells transfected with si-EZH2. D, PVT1 expression levels in cell cytoplasm or nucleus ofNSCLC cell lines A549 and PC-9 were detected by qPCR. GAPDH was used as a cytosol marker, and U1 was used as a nucleus marker. � , P < 0.05;�� , P < 0.01.






Figure 6.PVT1 could recruit PRC2 to LATS2 promoter and represses LATS2 transcription. A, RIPwith rabbitmonoclonal anti-EZH2, preimmune IgG, or 10% input fromA549 andPC-9 cell extracts. RNA levels in immunoprecipitates were detected by qPCR. Expression levels of PVT1 RNA are presented as fold enrichment in EZH2relative to IgG immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates were used as positive control. B, biotinylatedPVT1-positive RNA and negative RNA were incubated with A549 and PC-9 cell extracts. The RNA–protein complex was isolated from magnetic beadsbyBiotin ElutionBuffer.Western blotting assaywas conducted, and the results revealed that EZH2proteinwas pulled downbyPVT1. HURprotein is shownaspositivecontrol. C, RNA pulldown and Western blotting assays were performed, and the results revealed that 1 to 400 bp region of PVT1 could bind to EZH2. D,ChIP–qPCRof EZH2occupancy andH3K27-3mebinding in the LATS2promoter inA549 and PC-9 cells, and IgG as a negative control. E, at 48 hours after transfection,ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the LATS2 promoter in A549 and PC-9 cells treated with si-PVT1 or scrambled siRNA. F, therelationship betweenPVT1 expression and LATS2mRNA levelswas analyzed in the profile of NSCLCpatient tissue fromGEO. Themeanvalues andSDwere calculatedfrom triplicates of a representative experiment.

Wan et al.





associatedwith advanced pathologic stage and tumor size. Impor-tantly, PVT1 overexpression indicated poor prognosis and couldbe an independent prognostic indicator. In addition, PVT1 knock-down markedly inhibited NSCLC cell proliferation and inducedapoptosis both in vitro and in vivo. These data indicated that PVT1may serve as an oncogene and play a crucial role in NSCLCdevelopment and progression. Increasing evidence demonstratedthat lncRNAs regulate target genes via binding to the PRC2proteincomplex, and PRC2-mediated epigenetic regulation plays a keyrole in carcinogenesis. We found that lncRNA PVT1 is mostlylocated in the nuclear fractions in NSCLC cells, and RIP assaysrevealed that PVT1 could directly bind to EZH2, a core subunit ofthe PRC2 complex. We also found that knockdown of PVT1expression upregulated LATS2 expression in NSCLC cells, indi-cating that LATS2 may be an important underlying regulatorinvolved in PVT1 function.

LATS2, a member of the LATS tumor suppressor family,encodes a serine/threonine protein kinase (36). Accumulatingevidence indicates that LATS2 could be a new regulator of cellularhomeostasis, and downregulation of LATS2 has been shown inmany cancers, such as colorectal cancer and malignant mesothe-lioma (37, 38). Overexpression of LATS2 could inhibit thegrowth and motility of NSCLC cells (32). Furthermore, ourresults showed that ectopic expression of LATS2 also couldinduce G0-G1 phase arrest and cell apoptosis in NSCLC. Our

results also showed that MDM2 expression was decreased andp53was increasedwhen LATS2was overexpressed inNSCLC cells,which was consistent with treatment of si-PVT1 in NSCLC cells.These findings suggest that LATS2 could promote p53 activationby preventing MDM2-driven p53 degradation, which resulted ininduction of cell death.

Recent studies showed that the hypermethylation of the pro-moter regions of LATS2 likely plays an important role in thedownregulation of its mRNA expression in breast cancer andastrocytoma (39, 40). Moreover, many miRNAs, such as miR-25, miR-181b, and miR-93, suppressed LATS2 at the posttran-scription level by directly binding to its mRNA in multiple cancercells (41–43). Our results demonstrated that LATS2 could beregulated at the transcriptional level through histone modifica-tion mediated by lncRNA PVT1 and PRC2. Our ChIP assaysvalidated that EZH2 could directly bind to the promoter of LATS2in NSCLC cells, and knockdown of PVT1 led to loss of H3K27trimethylation and EZH2 binding ability to the promoter ofLATS2, confirming that LATS2 is direct target of PVT1/PRC2-regulated genes.

LncRNAs can function as oncogenes or tumor suppressor genesand regulate target gene expression at numerous levels, includingtranscriptional and posttranscriptional processing (44). LncRNACCAT1 promotes gallbladder cancer development through com-petitive "sponging" of miRNA-218-5p. LncRNA UFC1 interacts

Figure 7.Effect of LATS2 of overexpression onA549 cell in vitro.A, themRNA levels of LATS2 in A549 cells transfectedwith pCDNA–LATS2were detected by qPCR analysis. Band C, MTT and colony-forming assays were used to determine the cell viability for pCDNA–LATS2-transfected A549 cells. Values represent the mean� SD from three independent experiments. D, cell cycle was analyzed by flow cytometry. The bar chart represents the percentage of cells in G0–G1, S, or G2–Mphase,as indicated. E, apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. F, the protein level ofMdm2 and P53 in A549 cells transfected with pCDNA–LATS2 or si-PVT1was detected byWestern blot assays. All experiments were conducted in biologic triplicateswith three technical replicates. � , P < 0.05; �� , P < 0.01.






with the mRNA stabilizing protein HuR to regulate levels ofb-catenin, and promotes proliferation and reduces apoptosis inHCC cells (45, 46). Here, we found that PVT1 functioned as anoncogene and may be a crucial prognostic factor for NSCLCpatients. PVT1 contributed to lung adenocarcinoma cell prolif-eration, partly though EZH2-medicated suppression of the

LATS2/MDM2/P53 pathway. Our study may provide a novelstrategy for targeting the PVT1/EZH2/LATS2 axis as a new ther-apeutic application for NSCLC patients.

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

Figure 8.PVT1 negatively regulates expression of LATS2 by rescue assays. A, the levels of LATS2 mRNA and protein expression were determined by qPCR and Westernblotting assays when A549 cells were transfected with si-NC and si-PVT1 and cotransfected with si-PVT1 and si-LATS2. B and C, MTT and colony formationassays were used to determine the cell proliferation ability for A549 cells transfected with si-NC and si-PVT1 and cotransfected with si-PVT1 and si-LATS2. Valuesrepresent the mean � SD from three independent experiments. D and E, flow cytometry assays were performed to analyze the cell cycle and apoptosiswhen A549 cells were transfected with si-NC and si-PVT1 and cotransfected with si-PVT1 and si-LATS2. � , P < 0.05; �� , P < 0.01.

Wan et al.





Authors' ContributionsConception and design: L. WanZ.-X. WangDevelopment of methodology: G.-J. Liu, C.-C. WeiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Wan, G.-J. Liu, M.-D. HuangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. SunWriting, review, and/or revisionof themanuscript: L.Wan,M. Sun, Z.-X.WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.-B. Zhang, R. Kong, T.-P. XuStudy supervision: M. Sun

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China No. 81272601 and No. 81472198 (to Z.-X. Wang),

the Key Clinical Medicine Technology Foundation of Jiangsu ProvinceNo. BL2014096 (to Z.-X. Wang), the Medical Key Talented PersonFoundation of the Jiangsu Provincial Developing Health Project No.RC2011080 (to Z.-X. Wang), Innovation Team Project of the SecondAffiliated Hospital of Nanjing Medical University No. CX201202, "333High Class Talented Man Project" No. 2011-III-2630 (to Z.-X. Wang), andthe School Key Fund of Nanjing Medical University 2013NJMU054(to G.-J. Liu).

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 August 26, 2015; revised December 29, 2015; accepted January 27,2016; published OnlineFirst February 23, 2016.

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2016;15:1082-1094. Published OnlineFirst February 23, 2016.Mol Cancer Ther Li Wan, Ming Sun, Guo-Jian Liu, et al. ExpressionCell Proliferation through Epigenetically Regulating LATS2

Small Cell Lung Cancer− Promotes NonPVT1Long Noncoding RNA

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Long Noncoding RNA PVT1 Promotes Non Small Cell Lung … · NOP2 nucleolar protein (NOP2; refs.18, 19). In our previous study, we also found that increased PVT1 expression promoted