Long Noncoding RNA PVT1 Promotes Non–Small Cell Lung Cancer Cell

Published OnlineFirst February 23, 2016; DOI: 10.1158/1535-7163.MCT-15-0707

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Long Noncoding RNA PVT1 Promotes Non–Small Cell Lung Cancer Cell Proliferation through Epigenetically Regulating LATS2 Expression Li 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- well as poorer overall survival. Functional analysis revealed that scripts with no protein coding capacity, but with diverse func- knockdown of PVT1 inhibited NSCLC cell proliferation and tions in cancer cell proliferation, apoptosis, and metastasis. The induced apoptosis both in vitro and in vivo. RNA immunopre- lncRNA PVT1 is 1,716 nt in length and located in the chr8q24.21 cipitation and chromatin immunoprecipitation assays demon- region, which also contains the myelocytomatosis (MYC) onco- strated that PVT1 recruits EZH2 to the large tumor suppressor gene. Previous studies demonstrated that MYC promotes PVT1 kinase 2 (LATS2) promoter and represses LATS2 transcription. expression in primary human cancers. However, the expression Furthermore, ectopic expression of LATS2 increased apoptosis pattern and potential biologic function of PVT1 in non–small and repressed lung adenocarcinoma cell proliferation by regu- cell lung cancer (NSCLC) is still unclear. Here, we found that lating the Mdm2-p53 pathway. Taken together, our ﬁndings PVT1 was upregulated in 105 human NSCLC tissues compared indicated that PVT1/EZH2/LATS2 interactions might serve as with normal samples. High expression of PVT1 was associated new target for lung adenocarcinoma diagnosis and therapy. with a higher tumor–node–metastasis stage and tumor size, as Mol Cancer Ther; 15(5); 1082–94. 2016 AACR.

Introduction The rapid development in sequencing techniques and improve- ment in bioinformatics has led to the discovery of Long noncoding Lung cancer is a predominant cause of cancer-related death RNA (lncRNA), which are more than 200 bp in length and widely worldwide, due to its high morbidity and the lack of effective transcribed across the eukaryotic genome (4, 5). LncRNAs were therapy strategies (1). Non–small cell lung cancer (NSCLC) once regarded as transcriptional "noise" or cloning artifacts because accounts for approximately 85% of all lung cancer cases (2). of their absence of protein-encoding ability (6). Emerging evidence Despite recent advancements in clinical and experimental oncol- has indicated that aberrant expression of lncRNAs is associated ogy, the prognosis of NSCLC remains poor, with a 5-year overall with numerous diseases, including diabetes (7, 8). Importantly, survival rate of around 11% (3). Because NSCLC is a multigene lncRNAs were shown to act as regulators of tumorigenesis via disorder, the biologic processes involved in NSCLC tumorigenesis participating in biologic processes, such as modulation of apopto- are complicated. Thus, a detailed understanding of the mechan- sis, proliferation, drug resistance, and the epithelial–mesenchymal isms and molecular pathways underlying NSCLC development transition process (9–12). For example, our previous study dem- and progression is essential to improve diagnosis, prevention, and onstrated that the lncRNA Hox transcript antisense intergenic RNA treatment of NSCLC patients. (HOTAIR) contributes to cisplatin resistance of human lung adenocarcinoma cells via downregulation of p21WAF1/CIP1 expression. However, the contributions of aberrant lncRNAs to NSCLC devel- 1Department of Oncology, Second Affiliated Hospital, Nanjing Medical opment and progression and the underlying molecular pathways 2 University, Nanjing, People's Republic of China. Department of or mechanisms are not well documented. Oncology, Huai'an First People's Hospital, Nanjing Medical University, Huai'an, People's Republic of China. 3Department of Biochemistry and Chromatin-modifying complexes play important roles in Molecular Biology, Nanjing Medical University, Nanjing, People's lncRNA-mediated epigenetic regulation of gene expression. 4 Republic of China. Department of Oncology, First Affiliated Hospital, Recent studies showed that 24% of lncRNAs, one category of Nanjing Medical University, Nanjing, People's Republic of China. lncRNAs, are expressed in various cell types and bound by poly- Note: Supplementary data for this article are available at Molecular Cancer comb repressive complex 2 (PRC2), which comprises EZH2, Therapeutics Online (http://mct.aacrjournals.org/). SUZ12, and EED and acts as a molecular scaffold for histone L. Wan, M. Sun, and G.-J. Liu contributed equally to this article and should be modification complexes (13, 14). These lncRNAs recruit PRC2 to regarded as joint first authors. the promoter of target genes and catalyze the trimethylation of Corresponding Author: Zhao-Xia Wang, Second Affiliated Hospital, Nanjing lysine residue 27 of histone 3 (H3K27me3). For example, RASSF1 Medical University, 121 Jiangjiayuan Road, Nanjing 210011, People's Republic of antisense 1 (ANRASSF1) recruits PRC2 to the RASSF1A promoter, China. Phone: 025-58509810; Fax: 25-58509994; E-mail: resulting in silenced expression of RASSF1A and increased cell [email protected] proliferation. Ubiquitin-conjugating enzyme E2K (UBE2K) acts as doi: 10.1158/1535-7163.MCT-15-0707 a negative prognostic factor for lymph node metastasis and 2016 American Association for Cancer Research. survival via associating with PRC2 in bladder cancer (15, 16).

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PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

These data suggest that the dysregulation of PRC2-related Cell lines lncRNAs is involved in carcinogenesis. We purchased six NSCLC cell lines (PC-9, SPC-A1, H157, PVT1, an lncRNA that is 1,716 nt in length, is located in the H1299, A549, and SK-MES-1) and the normal bronchial epi- chr8q24.21 region which also contains the myelocytomatosis thelial cell line 16HBE from the Institute of Biochemistry and (MYC) oncogene. Interestingly, MYC protein leads to the accu- Cell Biology of the Chinese Academy of Sciences (Shanghai, mulation of PVT1 in primary human cancers (17). In addition, China). A549 and H1299 cells were cultured in RPMI-1640, high expression of PVT1 is a prognostic indicator for colorectal and 16HBE, PC-9, SPC-A1, H157, and SK-MES-1 cells were cancer patients and suppresses colorectal cancer cell apoptotic cultured in DMEM medium (GIBCO-BRL) supplemented with ability. Moreover, PVT1 could promote the proliferation and stem 10% FBS (Gibco),100 U/mL penicillin sodium, and 100 mg/ cell–like property of hepatocellular carcinoma cells by stabilizing mL streptomycin sulfate at 37Cinahumidified air atmosphere NOP2 nucleolar protein (NOP2; refs.18, 19). In our previous containing 5% CO2. All cell lines were authenticated by short study, we also found that increased PVT1 expression promoted tandem repeat DNA profiling. cell proliferation through binding with EZH2 and repressing P15 and P16 transcription in gastric cancer (20). Although Yang and RNA extraction and qPCR assays colleagues have demonstrated that PVT1 may serve as an "onco- TRIZOL reagent was used to extract total RNA from tissue gene" in NSCLC cells (21), but the potential molecular mechan- samples or cells according to the manufacturer's instructions. isms and downstream pathways of PVT1 in NSCLC cells are still Total RNA (500 ng) was reverse transcribed in a final volume of unknown. In this study, we investigated PVT1 expression in 10 mL using random primers under standard conditions with the NSCLC tissues and cell lines, and explored its biologic effects PrimeScript RT Reagent Kit (TaKaRa).To measure PVT1 expression and mechanisms in NSCLC cells in vitro and in vivo. levels, we used SYBR Premix Ex Taq (TaKaRa) following the manufacturer's instructions. GAPDH expression was used for Materials and Methods normalization. Primer sequences are listed in Supplementary Table S1. Real-time PCR was performed in triplicate on an ABI Tissue specimens and clinical data collection 7500, and data were calculated using the comparative cycle We obtained 105 paired NSCLC and adjacent nontumor lung DD threshold (CT) (2 CT) method. tissues from patients who underwent surgery at The First and fi Second Af liated Hospital of Nanjing Medical University from RNA interference 2010 to 2011. The patients were diagnosed with NSCLC (stages I, A549 and PC-9 cells were transfected with siRNA using Lipo- II, and III) based on histopathologic evaluation. The clinicopath- fectamine 2000 (Invitrogen), and the cells were incubated for 48 ologic characteristics of the NSCLC patients are summarized hours before use in assays. The siRNA sequences are listed in in Table 1. The patients did not receive any local or systemic Supplementary Table S1. The shRNA PVT1 (CCCAACAGGAG- treatment before operation. All collected tissue samples were GACAGCTT) was cloned into the pENTR/U6 vector. immediately snap-frozen in liquid nitrogen and stored at 80C until RNA extraction. The ethics committee of The Second Affil- Plasmid generation iated Hospital of Nanjing Medical University approved the study The LATS2 sequence was synthesized and subcloned into the protocol. pCDNA3.1 vector (GENECHEM) to generate the pCDNA–LATS2 vector for ectopic expression in cells. pCDNA3.1 vector was used Table 1. Correlation between PVT1 expression and clinicopathologic as a control. The qPCR assay was conducted to evaluate expression characteristics of NSCLC patients (n ¼ 105) of LATS2. We synthesized 1 to 400 bp, 1 to 800 bp, and 1 to 1,200 PVT1 Low number High number x2 test bp regions of PVT1 and subcloned into the pCDN3.1 vector, Characteristics of case (%) of case (%) (P value) respectively (Real Gene). Age (years) >65 27 (55.1) 28 (50.0) 0.696 Cell viability assays 65 22 (44.9) 28 (50.0) Gender The Cell Proliferation Reagent Kit I (MTT; Roche Applied Male 32 (65.3) 30 (53.6) 0.24 Science) was used to test the cell viability. A549 and PC-9 cells Female 17 (34.7) 26 (46.4) plated in 96-well plates (3,000 cells/well) were transfected with si- Histologic subtype PVT1. We estimated the cell viability every 24 hours according to Squamous cell carcinoma 28 (57.1) 35 (62.5) 0.69 the manufacturer's protocol. The MTT experiments were con- Adenocarcinoma 21 (42.9) 21 (37.5) ducted in quadruplicate. For colony formation assay, transfected TNM stage n ¼ Ia þ Ib 22 (44.9) 8 (14.3) 0.001a cells ( 1200) were placed in 6-well plates and maintained in IIa þ IIb 16 (32.7) 19 (33.9) proper medium containing 10% FBS for 2 weeks. The medium IIIa 11 (22.4) 29 (51.8) was replaced every 4 days, and visible colonies were counted after Tumor size 2 weeks. 5 cm 32 (65.3) 20 (35.7) 0.003a >5 cm 17 (34.7) 36 (64.3) Lymph node metastasis Flow cytometric analysis Negative 30 (61.2) 20 (35.7) 0.011a We harvested A549 and PC-9 cells that were transfected with Positive 19 (38.8) 36 (64.3) si-PVT1 for 48 hours. Double staining with FITC–Annexin V Smoking history and propidium iodide was performed using the FITC Annexin V Smokers 36 (73.5) 33 (58.9) 0.087 Apoptosis Detection Kit (BD Biosciences) according to the Never smokers 13 (26.5) 23 (41.1) manufacturer's recommendations. Cells were discriminated a Overall P < 0.05. into viable cells, dead cells, early apoptotic cells, and apoptotic

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cells, and the relative ratio of early apoptotic cells was com- of whole-cell extracts were incubated with treated beads for pared with control transfection from each experiment. For cell- 6 hours at 4C. We used the wash buffer to wash the beads for cycle analysis, cells were stained with propidium oxide using 6 times. To isolate the RNA–protein complexes from beads, the the CycleTEST PLUS DNA Reagent Kit (BD Biosciences) fol- beads were incubated with 0.1% SDS/0.5 mg/mL Proteinase K lowing the manufacturer's protocol and analyzed by FACScan. for 30 minutes at 55C. The qPCR assays were used to further The percentages of cells in G0–G1,S,andG2–M phases were evaluate the PRC2 isolated from the IP materials. counted and compared. Chromatin immunoprecipitation assays Western blot assay and antibodies Chromatin immunoprecipitation (ChIP) assays were con- Protein lysates were separated by 15% SDS-PAGE and trans- ducted using the EZ-CHIP KIT according to the manufacturer's m ferred to 0.22- m NC membranes (Sigma) and incubated with instructions (Millipore). EZH2 antibody was obtained from fi speci c primary antibodies. P53 and Mdm2 antibodies (1:1,000) Abcam. H3 trimethyl Lys 27 antibody was obtained from Milli- were purchased from SAB, Inc. Anti-LATS2 (1:1,000) was pur- pore. The ChIP primer sequences are listed in Supplementary chased from Abcam. GAPDH antibody was used as a control. Table S1. Quantification of immunoprecipitated DNA was performed using qPCR with SYBR Green Mix (TaKaRa). EZH2 could lncRNA expression from GEO DataSets bind to the 366 region of LATS2 promoter. ChIP data were NSCLC gene expression data were obtained from GEO DataSets calculated as a percentage relative to the input DNA. (GDS). Four independent data sets from GSE18842 (22), GSE19188 (23), GSE19804 (24), and GSE30219 (25) were RNA pulldown assays included in this study. The raw CEL files were downloaded from The pCDNA3.1-PVT1 vector was cut by restriction enzymes GEO database and normalized using Robust Multichip Average Nru I and treated with RNase-free Dnase I (Biolabs). LncRNA- (RMA). After we downloaded probe sequences from GEO or PVT1 was transcribed from cutted vector by mMESSAGE microarray manufacturers, blastþ2.2.30 was used to reannotates mMACHINE T7 Kit (Ambion) and purified with an RNeasy probe on GENCODE Release 21 sequence databases for lncRNA 0 Mini kit (Qiagen) in vitro. We biotin labeled the 3 end of and mRNA (26). For multiple probes corresponding to one gene, lncRNA-PVT1 referencing to the instruction of Pierce RNA 3 maximum normalized signal was selected to generate expressions End Desthiobiotinylation Kit (Thermo Scientific). One milli- of lncRNA and mRNA. Two-sample t test or paired-sample t test gram of protein from A549 and PC-9 cell extracts were then according to experimental design was employed as differential mixed with 50 pmol of biotinylated RNA, incubated with 50 expression calling method, followed by the Benjamini–Hochberg mL of magnetic beads for 1 hour at 4 C(ThermoScientific). (FDR) adjustment. For verifying expression correlation between The RNA–protein complex was isolated from magnetic beads genes, Pearson correlation analysis was used after CEL files from by Biotin Elution Buffer and boiled in SDS buffer for 10 GSE43580 (27) and GSE31210 (28) were downloaded and minutes. The retrieved protein was detected using the standard normalized by RMA. Western blot technique.

Tumor formation assay in a nude mouse model Immunohistochemistry Male athymic BALB/c mice (5-week-old) were maintained The primary tumors were immunostained for Ki-67 as previ- under specific pathogen-free conditions and manipulated accord- ously described (29). ing to protocols approved by the Shanghai Medical Experimental Animal Care Commission. A549 cells stably transfected with sh- PVT1 or empty vector were harvested at a concentration of 2 107 Statistical analysis cells/mL, and 0.1 mL was subcutaneously injected into the flanks All statistical analyses were performed using SPSS 20.0 fi of the nude mice, at one injection per mouse. Tumor growth was software (IBM). The signi cance of differences between t monitored, and tumor sizes and weights were measured every 3 groups was estimated by the Student test, Wilcoxon test, c2 days. Tumor volume was calculated using the formula, volume ¼ or test. Disease-free survival and overall survival rates were – (length width2 0.5). At 21 days after injection, the mice were calculated by the Kaplan Meier method with the log-rank test killed and tumor weights were measured. The primary tumors applied for comparison. The date of survival was evaluated by were excised, and tumor tissues were used for qRT-PCR analysis of univariate and multivariate Cox proportional hazards mod- P < PVT1 expression levels and immunostaining analysis of Ki-67 els. Variables with 0.05 in univariate analysis were used in protein expression. subsequent multivariate analysis on the basis of Cox regression analyses. Kendall Tau-b and Pearson correlation analyses PVT1 Subcellular fractionation were conducted to investigate the correlation between P The separation of nuclear and cytosolic fractions was per- and LATS2 expressions. Two-sided values were calculated, formed using the PARIS Kit (Life Technologies) according to the and a probability level of 0.05 was chosen for statistical fi manufacturer's instructions. signi cance.

RNA immunoprecipitation assays Results A549 and PC-9 cells were lysed for immunoprecipitation (IP) PVT1 expression is upregulated and correlated with a poor of endogenous PRC2 complexes from whole-cell extracts. The prognosis of NSCLC protein A Sepharose beads were incubated with positive anti- First, we analyzed the proﬁles of NSCLC patient from Gene body SNRNP70, negative antibody IgG, and interested antibody Expression Omnibus (GEO) and found that PVT1 was upre- EZH2 (Milipore) for 30 minutes at 4C. Then, the supernatants gulated in NSCLC tissues compared with normal lung tissues

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PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

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, relative expression of PVT1 in NSCLC tissues (n ¼ 105) and in paired adjacent normal tissues (n ¼ 105). PVT1 expression was examined by qPCR assays and normalized to GAPDH expression. Results are presented as the fold change in tumor tissues relative to normal tissues. C, the patients were divided into two groups according 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.

(Fig. 1A). To explore whether PVT1 was differently expressed in Association between PVT1 expression and patient survival NSCLC tissues, a total of 105 paired NSCLC tissue samples and Next, we conducted a Kaplan–Meier survival analysis to explore adjacent normal counterparts were evaluated for PVT1 expres- the correlation between PVT1 expression and NSCLC patient sion using qPCR assay. The results revealed that PVT1 expres- prognosis. Progression-free survival was 38.2% for the low PVT1 sion was upregulated in 75 tumor tissues (Fig. 1B). We used the group, and 17.0% for the high PVT1 group. Median survival time median expression of PVT1 as a cutoff point to divide all for the low PVT1 group was 31 months, and 15 months for the high patients into two groups: the high PVT1 group (n ¼ 56, fold PVT1 group (Fig. 1D). As shown in Fig. 1E, the overall survival rate change 3.0) and the low PVT1 group (n ¼ 49, fold change over 3 years for the low PVT1 group was 44.5%, and 25.7% for the 3.0). Statistical analysis revealed that PVT1 expression levels high PVT1 group. The median survival time for the low PVT1 group in NSCLC were signiﬁcantly correlated with tumor size (P ¼ was 33 months, and 18 months for the high PVT1 group. 0.003), advanced pathologic stage (P ¼ 0.001), and lymph Univariate survival analysis showed that lymph node metas- node metastasis (P ¼ 0.011). However, PVT1 expression was tasis, tumor–node–metastasis (TNM) stage, and PVT1 expression not associated with other factors including sex (P ¼ 0.24) and level could be viewed as prognostic factors (Table 2). Other age (P ¼ 0.696) in NSCLC (Table 1). clinicopathologic features, including sex and age, were not

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Table 2. Univariate and multivariate analysis of overall survival in NSCLC patients (n ¼ 105) Univariate analysis Multivariate analysis Variables HR (95% CI) P value HR (95% CI) P value Age 1.375 (0.753–2.510) 0.299 Gender 1.238 (0.679–2.259)) 0.486 Smoker 1.344 (0.736–2.453) 0.336 Histologic subtype 0.938 (0.513–1.717) 0.837 Chemotherapy 0.739 (0.425–1.287) 0.285 Tumor size 1.742 (0.947–3.203) 0.074 Lymph node metastasis (no vs. yes) 2.042 (1.105–3.776) 0.023a 1.429 (0.739–2.764) 0.288 TNM 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.

statistically signiﬁcant prognostic factors. Moreover, multivariate cell lines (from The Cancer Genome Atlas data) and NSCLC Cox regression analyses showed that expression of PVT1 tissues (GSE28571 and GSE43850). We found that the PVT1 (P ¼ 0.012), along with TNM stage (P ¼ 0.024), was an inde- expression level is positively related with MYC expression both pendent prognostic factor for NSCLC patients (Table 2). in cells and tissues (Supplementary Fig. S1). We next investigated the biologic effect of PVT1 on NSCLC cells. Knockdown of PVT1 inhibits NSCLC cell proliferation, induces First, a qPCR assay was performed to evaluate the expression of apoptosis, and promotes cell-cycle arrest PVT1 in various NSCLC cell lines. As shown in Fig. 2A, four cell Previous study showed that MYC protein could lead to the lines (A549, PC-9, SK-MES-1, and H157) expressed higher levels accumulation of PVT1 in primary human cancers; hence, we of PVT1 compared with the normal bronchial epithelial cell line analyzed the relationship between PVT1 and MYC in lung cancer (16HBE). In contrast, the relative expression level of PVT1 was

Figure 2. Effects of knockdown of PVT1 on NSCLC cell viability in vitro.A,PVT1 expression levels of NSCLC cell lines (A549, PC-9, SK-MES-1, H157, SPCA1, and H1299) compared with that in normal human bronchial epithelial cells (16HBE). B, A549 and PC-9 cells were transfected with si-PVT1. C, MTT assays were conducted to determine the cell proliferation ability for si-PVT1– transfected A549 and PC-9 cells. Values indicate the mean SD from three independent experiments. D, colony- forming assays were performed to determine the proliferation of si-PVT1–transfected A549 and PC-9 cells. , P < 0.05; , P < 0.01; NS, not signiﬁcant.

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PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

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 the percentage of cells in G0-G1,S,orG2–M phase, as indicated. C and D, A549 and PC-9 cells were stained and analyzed by ﬂow cytometry. LR, early apoptotic cells; 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.

lower in H1299 cell lines. Next, we knocked down endogenous PVT1 was significantly lower than that in the control group (Fig. PVT1 expression in A549 and PC-9 cells by siRNAs. At 48 hours 4C). Next, we used a qPCR assay to explore the average after transfection, PVT1 expression was reduced by approximately expression of PVT1 in tumor tissues (Fig. 4D). As shown 78.1% or 81.9% compared with control siRNA-transfected cells in Fig. 4E, immunohistochemistry (IHC) analysis confirmed (Fig. 2B). MTT and colony formation assays demonstrated that that the tumors formed from A549/sh-PVT1 cells displayed growth of A549 and PC-9 cells transfected with si-PVT1 was lower Ki-67 staining than those formed from the control cells. attenuated compared with control cells (Fig. 2C and D). Further- Our results indicated that knockdown of PVT1 expression could more, flow cytometry analysis revealed that knockdown of PVT1 suppress tumor growth in vivo. expression induced apoptosis and cell-cycle arrest at G1–G0 phase in A549 and PC-9 cells (Fig. 3A–D). LATS2 is a key downstream mediator of PVT1 To explore the underlying target genes of PVT1 in NSCLC, we Knockdown of PVT1 inhibits NSCLC cell tumorigenesis analyzed previously published gene expression profile down- in vivo stream of PVT1 in colorectal cancer cells by Takahashi and To further investigate whether reduction of PVT1 expression colleagues (18).The qPCR assay was performed to detect the could impact tumor growth in vivo, A549 cells stably transfected expression of the cyclin B1 (CCNB1), ankyrin repeat and GTPase with sh-PVT1 or empty vectors were inoculated into male nude domain Arf GTPase activating protein 11 (ACAP11), SMAD family mice. Eighteen days after injection, the tumor size in the sh- member 4 (SMAD4), which were mentioned in the earlier study. PVT1 group was decreased substantially compared with the Unexpectedly, the qPCR results showed that PVT1 knockdown control group (Fig. 4A and B). The mean tumor weight of sh- did not affect the expression of these genes in A549 and PC-9 cells,

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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 male nude mice (n ¼ 6), respectively. Tumor volumes were calculated after injection every 3 days. Bars, SD. C, tumor weights are represented as mean of tumor 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 under hematoxylin and eosin (H&E) staining and IHC staining using antibodies against Ki-67. , P < 0.01.

but increased the expression of LATS2 (Fig. 5A). To independently cipitates. Furthermore, we conducted RNA pulldown assays verify this result, we conducted Western blot analysis to measure in A549 and PC-9 cells to determine whether EZH2 is LATS2 expression. The results revealed that LATS2 protein levels associated with PVT1. The results revealed that EZH2 could were also increased in si-PVT1–transfected cells (Fig. 5B). interact with PVT1 (Fig.6B).Moreover,wesynthesizedthree regions of PVT1, including 1 to 400 bp, 1 to 800 bp, and 1 PVT1 inhibits LATS2 expression by binding to EZH2 to 1,200 bp. Then, RNA pulldown assays were performed to Next, we investigated the mechanism by which PVT1 regu- confirm which region of PVT1 couldbindtoEZH2,andthe lates LATS2 expression. First, qPCR assays were performed to results showed that 1 to 400 bp of PVT1 is the major evaluate PVT1 expression in nuclear and cytosolic fractions binding site (Fig. 6C). from A549 and PC-9 cells. GAPDH and U1 RNA were used as LATS2 is a tumor suppressor gene that is mutated in NSCLC, fractionation indicators. The results demonstrated higher and downregulation of LATS2 could promote NSCLC cell expression of PVT1 in the nucleus versus the cytosol in both viability and motility (31, 32). A previous study showed that cell lines (Fig. 5D). hypermethylation of the promoter regions of LATS2 played a PVT1 might play a major regulatory function at the tran- vital role in the downregulation of its mRNA levels in NSCLC scriptional level. Approximately 24% of lncRNAs physically (33). EZH2, a component of PRC2, could mediate H3K27me3 associate with PRC2. Thus, we next evaluated whether PVT1 modification and epigenetically silence target genes. To further couldbindtothePRC2complex.RNA–protein interaction determine whether EZH2 can directly bind the promoter region prediction (RPISeq) analysis showed that the PVT1-EZH2 and of LATS2, we designed four pairs of primers across 1,000 bp of SUZ12-SUZ12 interaction scores were 0.87 and 0.83 with the promoter region. CHIP assays confirmed that EZH2 could Support Vector Machine classifier, respectively. Predictions bind to the LATS2 promoter region (Fig. 6D). Moreover, with probabilities >0.5 were considered positive, and accuracies knockdown of PVT1 reduced EZH2 binding to LATS2 promoter of the prediction ranged from 57% to 99% in independent regions (Fig. 6E). Finally, we analyzed the correlation between datasets of RNA–protein interactions (30). PVT1 and LATS2 expressions in NSCLC patient profiles from Next, RNA immunoprecipitation (RIP) experiments were GEO and found that there was a significantly negative corre- performed in A549 and PC-9 cell extracts using antibodies lation (Fig. 6F). against EZH2 and SUZ12. We used qPCR assay to detect RNA levels in immunoprecipitates. As shown in Fig. 6A, the LATS2 may be involved in the Mdm2-P53 pathway results revealed PVT1 enrichment in EZH2-RNA precipitates, To further investigate whether LATS2 is involved in the PVT1- whereas less enrichment was observed in SUZ12-RNA pre- induced increase of NSCLC cells apoptosis and cycle arrest, we

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PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

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 transfected with si-PVT1, and results are expressed relative to the corresponding values for control cells. B, Western blot assay was conducted to detect the level of LATS2 protein in A549 or PC-9 cells transfected with si-PVT1. C, Western blot and qPCR assays were used to detect the LATS2 expression both in mRNA and protein levels in A549 and PC-9 cells transfected with si-EZH2. D, PVT1 expression levels in cell cytoplasm or nucleus of NSCLC 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.

performed gain-of-function assays. The qPCR assay results con- C). Meanwhile, apoptosis rate and cycle arrest also were firmed that LATS2 expression was significantly upregulated in rescued in cotransfected group comparing with si-PVT1 group A549 cells transfected with pCDNA–LATS2 compared with the (Fig. 8D and E). These dates indicate that PVT1 exerting control group (Fig. 7A). MTT and colony formation assays dem- biologic effect on NSCLC cells may partly through repressing onstrated that the NSCLC cell viability was inhibited dramatically LATS2 expression. upon overexpression of LATS2 (Fig. 7B and C). Moreover, flow cytometry analysis showed a G0-G1 cell-cycle arrest and increased apoptosis rate in A549 cells transfected with pCDNA–LATS2 Discussion (Fig. 7D and E). A previous report showed that LATS2 could bind Recently, a large number of studies have demonstrated that Mdm2 to inhibit its E3 ligase activity and activate p53 (34).Thus, lncRNA dysregulation is involved in epigenetics and participates we performed Western blot analysis of Mdm2 and p53 expres- in carcinogenesis. For instance, lncRNA HOTAIR promotes gastric sions, and the results showed that Mdm2 expression was cancer metastasis through inhibition of Poly r(C) Binding Protein decreased and p53 expression was increased in A549 cells trans- (PCBP) 1 (35). In our previous studies, we found that p53- fected with pCDNA–LATS2. Cells with knocked down PVT1 regulated lncRNA TUG1 affected NSCLC cell proliferation via showed consistent results (Fig. 7F). Thus, these findings suggest epigenetically regulating HOXB7 expression. We also showed that that PVT1 could participate in the Mdm2-p53 pathway by epi- EZH2-mediated epigenetic suppression of lncRNA SPRY4-IT1 genetically regulating LATS2. promoted NSCLC cell proliferation and metastasis by affecting Moreover, we conducted rescue assays to determine whether the epithelial–mesenchymal transition. However, as the lncRNA PVT1 contributes to NSCLC cell proliferation and apoptosis research field has expanded quickly and more lncRNAs have been via inhibiting LATS2 expression. A549 cells were cotransfected characterized, important roles for aberrant lncRNAs in the devel- with si-PVT1 and si-LATS2.AsshowninFig.8A,si-LATS2 opment and metastasis of multiple cancers have been demon- transfection could partly rescue si-PVT1–increased LATS2 strated. Therefore, the contributions of misregulated lncRNAs to expression (Fig. 8A). The results of MTT and colony formation NSCLC, and their biologic function and underlying mechanism in assays indicated that the proliferation ability of A549 cells NSCLC cells should be investigated. cotransfected with si-PVT1 and si-LATS2 was improved com- In this study, we found that another lncRNA PVT1 is upregu- paring with A549 cells transfected with si-PVT1 (Fig. 8B and lated in NSCLC tissues, and increased PVT1 expression is

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Figure 6. PVT1 could recruit PRC2 to LATS2 promoter and represses LATS2 transcription. A, RIP with rabbit monoclonal anti-EZH2, preimmune IgG, or 10% input from A549 and PC-9 cell extracts. RNA levels in immunoprecipitates were detected by qPCR. Expression levels of PVT1 RNA are presented as fold enrichment in EZH2 relative to IgG immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates were used as positive control. B, biotinylated PVT1-positive RNA and negative RNA were incubated with A549 and PC-9 cell extracts. The RNA–protein complex was isolated from magnetic beads by Biotin Elution Buffer. Western blotting assay was conducted, and the results revealed that EZH2 protein was pulled down by PVT1. HUR protein is shown as positive control. 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–qPCR of EZH2 occupancy and H3K27-3me binding in the LATS2 promoter in A549 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, the relationship between PVT1 expression and LATS2 mRNA levels was analyzed in the proﬁle of NSCLC patient tissue from GEO. The mean values and SD were calculated from triplicates of a representative experiment.

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PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

Figure 7. Effect of LATS2 of overexpression on A549 cell in vitro. A, the mRNA levels of LATS2 in A549 cells transfected with pCDNA–LATS2 were detected by qPCR analysis. B and 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,orG2–M phase, as indicated. E, apoptosis was determined by ﬂow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. F, the protein level of Mdm2 and P53 in A549 cells transfected with pCDNA–LATS2 or si-PVT1 was detected by Western blot assays. All experiments were conducted in biologic triplicates with three technical replicates. , P < 0.05; , P < 0.01.

associated with advanced pathologic stage and tumor size. Impor- results also showed that MDM2 expression was decreased and tantly, PVT1 overexpression indicated poor prognosis and could p53 was increased when LATS2 was overexpressed in NSCLC cells, be an independent prognostic indicator. In addition, PVT1 knock- which was consistent with treatment of si-PVT1 in NSCLC cells. down markedly inhibited NSCLC cell proliferation and induced These findings suggest that LATS2 could promote p53 activation apoptosis both in vitro and in vivo. These data indicated that PVT1 by preventing MDM2-driven p53 degradation, which resulted in may serve as an oncogene and play a crucial role in NSCLC induction of cell death. development and progression. Increasing evidence demonstrated Recent studies showed that the hypermethylation of the pro- that lncRNAs regulate target genes via binding to the PRC2 protein moter regions of LATS2 likely plays an important role in the complex, and PRC2-mediated epigenetic regulation plays a key downregulation of its mRNA expression in breast cancer and role in carcinogenesis. We found that lncRNA PVT1 is mostly astrocytoma (39, 40). Moreover, many miRNAs, such as miR- located in the nuclear fractions in NSCLC cells, and RIP assays 25, miR-181b, and miR-93, suppressed LATS2 at the posttran- revealed that PVT1 could directly bind to EZH2, a core subunit of scription level by directly binding to its mRNA in multiple cancer the PRC2 complex. We also found that knockdown of PVT1 cells (41–43). Our results demonstrated that LATS2 could be expression upregulated LATS2 expression in NSCLC cells, indi- regulated at the transcriptional level through histone modifica- cating that LATS2 may be an important underlying regulator tion mediated by lncRNA PVT1 and PRC2. Our ChIP assays involved in PVT1 function. validated that EZH2 could directly bind to the promoter of LATS2 LATS2, a member of the LATS tumor suppressor family, in NSCLC cells, and knockdown of PVT1 led to loss of H3K27 encodes a serine/threonine protein kinase (36). Accumulating trimethylation and EZH2 binding ability to the promoter of evidence indicates that LATS2 could be a new regulator of cellular LATS2, confirming that LATS2 is direct target of PVT1/PRC2- homeostasis, and downregulation of LATS2 has been shown in regulated genes. many cancers, such as colorectal cancer and malignant mesothe- LncRNAs can function as oncogenes or tumor suppressor genes lioma (37, 38). Overexpression of LATS2 could inhibit the and regulate target gene expression at numerous levels, including growth and motility of NSCLC cells (32). Furthermore, our transcriptional and posttranscriptional processing (44). LncRNA results showed that ectopic expression of LATS2 also could CCAT1 promotes gallbladder cancer development through com- induce G0-G1 phase arrest and cell apoptosis in NSCLC. Our petitive "sponging" of miRNA-218-5p. LncRNA UFC1 interacts

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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 Western blotting 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 formation assays 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. Values represent the mean SD from three independent experiments. D and E, ﬂow cytometry assays were performed to analyze the cell cycle and apoptosis when A549 cells were transfected with si-NC and si-PVT1 and cotransfected with si-PVT1 and si-LATS2. , P < 0.05; , P < 0.01.

with the mRNA stabilizing protein HuR to regulate levels of LATS2/MDM2/P53 pathway. Our study may provide a novel b-catenin, and promotes proliferation and reduces apoptosis in strategy for targeting the PVT1/EZH2/LATS2 axis as a new ther- HCC cells (45, 46). Here, we found that PVT1 functioned as an apeutic application for NSCLC patients. oncogene and may be a crucial prognostic factor for NSCLC patients. PVT1 contributed to lung adenocarcinoma cell prolif- Disclosure of Potential Conﬂicts of Interest eration, partly though EZH2-medicated suppression of the No potential conﬂicts of interest were disclosed.

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PVT1 Promotes NSCLC Cell Proliferation by Regulating LATS2

Authors' Contributions the Key Clinical Medicine Technology Foundation of Jiangsu Province Conception and design: L. WanZ.-X. Wang No. BL2014096 (to Z.-X. Wang), the Medical Key Talented Person Development of methodology: G.-J. Liu, C.-C. Wei Foundation of the Jiangsu Provincial Developing Health Project No. Acquisition of data (provided animals, acquired and managed patients, RC2011080 (to Z.-X. Wang), Innovation Team Project of the Second ﬁ provided facilities, etc.): L. Wan, G.-J. Liu, M.-D. Huang Af liated Hospital of Nanjing Medical University No. CX201202, "333 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, High Class Talented Man Project" No. 2011-III-2630 (to Z.-X. Wang), and computational analysis): M. Sun the School Key Fund of Nanjing Medical University 2013NJMU054 Writing, review, and/or revision of the manuscript: L. Wan, M. Sun, Z.-X. Wang (to G.-J. Liu). Administrative, technical, or material support (i.e., reporting or organizing The costs of publication of this article were defrayed in part by the data, constructing databases): E.-B. Zhang, R. Kong, T.-P. Xu payment of page charges. This article must therefore be hereby marked advertisement Study supervision: M. Sun in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant Support This work was supported by grants from the National Natural Science Received August 26, 2015; revised December 29, 2015; accepted January 27, Foundation of China No. 81272601 and No. 81472198 (to Z.-X. Wang), 2016; published OnlineFirst February 23, 2016.

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Long Noncoding RNA PVT1 Promotes Non−Small Cell Lung Cancer Cell Proliferation through Epigenetically Regulating LATS2 Expression

Li Wan, Ming Sun, Guo-Jian Liu, et al.

Mol Cancer Ther 2016;15:1082-1094. Published OnlineFirst February 23, 2016.

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