longnoncodingrnaanrilpromotesnon small cell lung cancer...

Cancer Biology and Signal Transduction

LongNoncodingRNAANRIL PromotesNon–SmallCell Lung Cancer Cell Proliferation and InhibitsApoptosis by Silencing KLF2 and P21 ExpressionFeng-qi Nie1, Ming Sun2, Jin-song Yang3, Min Xie2, Tong-peng Xu1, Rui Xia2,Yan-wen Liu2,Xiang-hua Liu2, Er-bao Zhang2, Kai-hua Lu1, and Yong-qian Shu1

Abstract

Recent evidence highlights long noncoding RNAs (lncRNA)as crucial regulators of cancer biology that contribute to essen-tial cancer cell functions such as cell proliferation, apoptosis,and metastasis. In non–small cell lung cancer (NSCLC), severallncRNAs' expressions are misregulated and have been nomi-nated as critical actors in NSCLC tumorigenesis. LncRNA ANRILwas first found to be required for the PRC2 recruitment to andsilencing of p15INK4B, the expression of which is induced by theATM–E2F1 signaling pathway. Our previous study showed thatANRIL was significantly upregulated in gastric cancer, and itcould promote cell proliferation and inhibit cell apoptosis bysilencing of miR99a and miR449a transcription. However, itsclinical significance and potential role in NSCLC is still notdocumented. In this study, we reported that ANRIL expression

was increased in NSCLC tissues, and its expression level wassignificantly correlated with tumor–node–metastasis stages andtumor size. Moreover, patients with high levels of ANRILexpression had a relatively poor prognosis. In addition, takingadvantage of loss-of-function experiments in NSCLC cells, wefound that knockdown of ANRIL expression could impair cellproliferation and induce cell apoptosis both in vitro and vivo.Furthermore, we uncover that ANRIL could not repress p15expression in PC9 cells, but through silencing of KLF2 and P21transcription. Thus, we conclusively demonstrate that lncRNAANRIL plays a key role in NSCLC development by associatingits expression with survival in patients with NSCLC, providingnovel insights on the function of lncRNA-driven tumorigenesis.Mol Cancer Ther; 14(1); 268–77. �2014 AACR.

IntroductionLung cancer is the most common type of cancer and the

primary cause of cancer-related death worldwide (1). Non–small cell lung cancer (NSCLC) accounts for 80% of all lungcancer cases, represents the most prevalent class of this cancertype, and includes several histologic subtypes such as squa-mous cell carcinoma (SCC), adenocarcinoma and large cellcarcinoma (LCC; refs. 2, 3). Despite current advances in thetreatments for NSCLC, including surgical therapy, chemother-apy, and molecular targeting therapy, the overall 5-year sur-vival rate for patients with NSCLC has not been markedlyimproved over years and remains as low as 15% (4). Therefore,a greater understanding of the molecular mechanisms involved

in the development, progression, and spread of the NSCLC isessential for the developing of specific diagnostic methods anddesigning of more individualized and effective therapeuticstrategies.

Recently, studies using the great advances in genomic tech-nologies have revealed the majority of the human genome istranscribed, whereas only 2% of the transcribed genome codesfor protein (5). Meanwhile, it is becoming increasingly appar-ent that the large majority of genome is transcribed intononcoding RNAs (ncRNAs), including microRNAs and longncRNAs (lncRNAs; ref. 6). The ENCODE Consortium haselucidated the prevalence of thousands of human lncRNAs,but only very few of them have been assigned with any biologicfunction (7). To date, studies showed that miRNAs play impor-tant roles in the posttranscriptional regulation of gene expres-sion; however, the lncRNAs counterpart of ncRNA is not wellcharacterized (8). Although very few are characterized in detail,LncRNAs are involved in a large range of biologic processes,including modulation of apoptosis and invasion, reprogram-ming stem cell pluripotency, and parental imprinting throughthe regulation of gene expression by chromatin remodeling,histone protein modification, regulation of mRNA splicing, andacting as sponges for microRNAs (9–12).

In the past decade, lots of evidence have linked the dysregula-tion of lncRNAswith diverse humandiseases, in particular cancers(13–15). Therefore, identification of cancer-associated lncRNAsand investigation of their molecular and biologic functions areimportant in understanding the molecular biology of NSCLCdevelopment and progression. Our previous study showed thatlncRNA ANRIL was significantly upregulated in gastric cancer,

1Department of Oncology, First Affiliated Hospital, Nanjing MedicalUniversity, Nanjing, People's Republic of China. 2Department of Bio-chemistry andMolecular Biology, NanjingMedical University, Nanjing,People's Republic of China. 3Department of Oncology, Nanjing FirstHospital, Nanjing Medical University, People's Republic of China.

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

F.-q. Nie, M. Sun, and J.-s. Yang contributed equally and are joint first authors tothis article.

Corresponding Authors: Kai-hua Lu, Department of Oncology, First AffiliatedHospital, Nanjing Medical University, Han Zhong Road 140#, Nanjing 210029,China. Phone/fax: 25-8686-2728; E-mail: [email protected]; and Yong-qian Shu,[email protected]

doi: 10.1158/1535-7163.MCT-14-0492

�2014 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 14(1) January 2015268

on February 3, 2019. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 12, 2014; DOI: 10.1158/1535-7163.MCT-14-0492

http://mct.aacrjournals.org/

and increased ANRIL promoted gastric cancer cells prolifera-tion and inhibited apoptosis by epigenetic silencing of miR99aand miR449a transcription (16). Moreover, ANRIL can bindto and recruits PRC2 to repress the expression of p15INK4B locus,which resulted in increased cell proliferation (17, 18). However,the ANRIL clinical significance and potential role in NSCLCdevelopment and progression is still not documented.

In this study, we found that lncRNA ANRIL expression wasincreased in NSCLC tissues compared with adjacent normaltissues. Its expression level was significantly correlated withtumor–node–metastasis (TNM) stages and tumor size. More-over, patients with higher level of ANRIL expression had arelatively poor prognosis. Furthermore, we investigated theeffects of ANRIL expression on NSCLC cell phenotype in vitroand in vivo with the loss-of-function study. Moreover, we alsoshowed that ANRIL could bind to PRC2 to repress KLF2 andP21 transcription, but not regulate P15INK4 expression inNSCLC PC9 cells, which indicated that ANRIL affected NSCLC

cells proliferation and apoptosis partly via silencing of KLF2and P21 transcription. This study advances our understandingof the role of lncRNAs, such as a regulator of pathogenesis ofNSCLC and facilitates the development of lncRNA-directeddiagnostics and therapeutics.

Materials and MethodsTissue collection

We obtained 68 paired NSCLC and adjacent nontumor lungtissues from patients who underwent surgery at Jiangsu ProvinceHospital between 2010 and 2011, and were diagnosed withNSCLC based on histopathologic evaluation. Clinicopathologiccharacteristics, including TNM staging, were recorded. No local orsystemic treatment was conducted in these patients before sur-gery. All collected tissue sampleswere immediately snap-frozen inliquid nitrogen and stored at�80�Cuntil required. Our studywasapproved by the Research Ethics Committee of Nanjing Medical

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Figure 1.Relative ANRIL expression in NSCLC tissues and its clinical significance. A, relative expression of ANRIL in NSCLC tissues (n ¼ 68) compared with correspondingnontumor tissues (n ¼ 68). ANRIL expression was examined by qPCR and normalized to GAPDH expression. Results are presented as the fold changein tumor tissues relative to normal tissues. B, ANRIL expression was classified into two groups. C and D, Kaplan–Meier DFS and OS curves according to ANRILexpression levels.

ANRIL Promotes Proliferation in NSCLC

www.aacrjournals.org Mol Cancer Ther; 14(1) January 2015 269




University, China. Written informed consent was obtained fromall patients.

Cell linesFive NSCLC adenocarcinoma cell lines (PC9, SPC-A1, NCI-

H1975, H1299, and H358), and one NSCLC squamous carcino-mas cell lines (H520) were purchased from the Institute ofBiochemistry and Cell Biology of the Chinese Academy ofSciences (Shanghai, China). A549, H1975, H1299, and H520cells were cultured in RPMI-1640; 16HBE, PC9, and SPC-A1 cellswere cultured in DMEM (GIBCO-BRL) medium supplementedwith 10%FBS, 100U/mLpenicillin and 100mg/mL streptomycin(Invitrogen, Carlsbad) at 37oC/5% CO2. All cell lines wereauthenticated by short tandem repeat DNA profiling.

RNA extraction and qPCR assaysTotal RNA was isolated with TRizol reagent (Invitrogen)

according to the manufacturer's instructions. Total RNA (500ng) was reverse transcribed in a final volume of 10 mL usingrandom primers under standard conditions for the PrimeScriptRT reagent Kit (TaKaRa, Dalian, China). We used the SYBRPremix Ex Taq (TaKaRa, Dalian, China) to determine ANRILexpression levels, following the manufacturer's instructions.Results were normalized to the expression of GAPDH. Thespecific primers used are shown in Supplementary Table S1.The qPCR assays were conducted on an ABI 7500, and datacollected with this instrument. Our qPCR results were analyzedand expressed relative to threshold cycle (Ct) values, and thenconverted to fold changes.

Cell transfectionHuman ANRIL cDNA clone L6ChoCKO-2-E10 with functional

region was provided by the Functional Genomics Research Cen-ter, KRIBB, Korea. Plasmid vectors (pCNS-ANRIL, sh-ANRIL andempty vector) for transfectionwere prepared usingDNAMidiprepor Midiprep kits (Qiagen), and transfected into16HBE, SPC-A1,H1299, or PC9cells. The si-ANRIL or si-NC was transfected intoSPC-A1, H1299, or PC9 cells. SPC-A1, H1299, or PC9 cells weregrown on 6-well plates to confluency and transfected usingLipofectamine 2000 (Invitrogen) according to themanufacturer'sinstructions. At 48hours after transfection, cellswereharvested forqPCR or Western blot analysis.

Cell viability assaysCell viability was monitored using a Cell Proliferation Reagent

Kit I (MTT; Roche Applied Science). The SPC-A1, H1299, PC9, orA549 cells transfectedwith si-ANRIL (3000 cells/well) were grownin 96-well plates. Cell viability was assessed every 24 hoursfollowing the manufacturer's protocol. All experiments wereperformed in quadruplicate. For each treatment group wells wereassessed in triplicate.

Flow cytometrySPC-A1, H1299, or PC9 cells transfected with si-ANRIL were

harvested 48 hours after transfection by trypsinization. After thedouble staining with FITC–Annexin V and Propidium iodide (PI)was done using the FITC–Annexin V Apoptosis Detection Kit (BDBiosciences) according to the manufacturer's recommendations,the cells were analyzed with a flow cytometry (FACScan; BDBiosciences) equipped with a CellQuest software (BD Bio-sciences). Cells were discriminated into viable cells, dead cells,

early apoptotic cells, and apoptotic cells, and then the relativeratio of early apoptotic cells were compared with control trans-fectant from each experiment. Cells for cell–cycle analysis werestained with PI using the CycleTEST PLUS DNA Reagent Kit (BDBiosciences) following the protocol and analyzed by FACScan.The percentage of the cells in G0–G1, S, and G2–M phase werecounted and compared.

Tumor formation assay in a nude mouse modelFemale athymic BALB/c nude mice (4-weeks-old) were main-

tained under pathogen-free conditions and manipulatedaccording to protocols approved by the Shanghai MedicalExperimental Animal Care Commission. PC9 cells were stablytransfected with sh-ANRIL and empty vector and harvestedfrom 6-well cell culture plates, washed with PBS, and resus-pended at a concentration of 1� 108 cells/mL. A total of 100 mLof suspended cells was s.c. injected into a single side of theposterior flank of each mouse. Tumor growth was examinedevery 3 days, and tumor volumes were calculated using theequation V ¼ 0.5 � D � d2 (V, volume; D, longitudinaldiameter; d, latitudinal diameter). At 18 days after injection,mice were euthanized, and the subcutaneous growth of eachtumor was examined. This study was carried out in strictaccordance with the recommendations in the Guide forthe Care and Use of Laboratory Animals of the NationalInstitutes of Health. The protocol was approved by the Com-mittee on the Ethics of Animal Experiments of the Nanjingmedical University.

RNA immunoprecipitationFor immunoprecipitation (IP) of endogenous PRC2 complexes

from whole-cell extracts, cells were lysed. The supernatants wereincubated with protein A Sepharose beads coated with antibodiesthat recognized EZH2, SNRNP70, or with control IgG (Millipore)

Table 1. Correlation between ANRIL expression and clinicopathologiccharacteristics of NSCLC patients

ANRIL

CharacteristicsHigh, number ofcases (34)

Low, number ofcases (34)

c2 test(P value)

Age, y 0.627�65 17 19>65 17 15

Gender 0.625Male 18 20Female 16 14

Histologic subtype 0.324SCC 22 18Adenocarcinoma 12 16

TNM stage 0.007a

Ia þ Ib 4 15IIa þ IIb 14 12IIIa 16 7

Tumor size 0.001a

�5 cm 13 26>5 cm 21 8

Lymph node metastasis 0.051Negative 11 19Positive 23 15

Smoking history 0.793Smokers 23 24Never smokers 11 10

aOverall P < 0.05.

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Mol Cancer Ther; 14(1) January 2015 Molecular Cancer Therapeutics270




for 6 hours at 4�C. After the beads were washed with wash buffer,the complexes were incubated with 0.1% SDS/0.5 mg/mL Pro-teinase K (30 minutes at 55�C) to remove proteins, respectively.The PRC2 isolated from the IP materials was further assessed byqPCR analysis (19).

Chromatin immunoprecipitationPC9 cells were treated with formaldehyde and incubated for

10 minutes to generate DNA–protein cross-links. Cell lysateswere then sonicated to generate chromatin fragments of 200 to300 bp and immunoprecipitated with EZH2 and H3K27me3-specific antibody (Cell Signaling Technology) or IgG as control.Precipitated chromatin DNA was recovered and analyzed byqPCR.

Western blot assay and antibodiesCells protein lysates were separated by 10% SDSPAGE,

transferred to 0.22 mm NC membranes (Sigma), and incubated

with specific antibodies. ECL chromogenic substrate was usedand quantified by densitometry (Quantity One software; Bio-Rad). GAPDH antibody was used as control, anti-P21, CDK2,CDK4, CDK6, P15, and PARP (1:1,000) were purchased fromCell Signaling Technology, Inc.; anti-KLF2 was purchased fromSigma.

Statistical analysisAll statistical analyseswere performed using SPSS 17.0 software

(IBM). The significance of differences between groups was esti-mated by the Student t test, Wilcoxon test, or c2 test. Disease-freesurvival (DFS) and overall survival (OS) rates were calculated bythe Kaplan–Meier method with the log-rank test applied forcomparison. The date of survival was evaluated by univariateand multivariate Cox proportional hazards models. Variableswith P < 0.05 in univariate analysis were used in subsequentmultivariate analysis on the basis of Cox regression analyses.Kendall Tau-b and Pearson correlation analyses were used to

Figure 2.Effects of knockdown of ANRIL on NSCLC cell viability in vitro. A, ANRIL expression levels of NSCLC cell lines (PC9, SPC-A1, NCI-H1975, H1299, and H358 andH520) compared with that in normal human bronchial epithelial cells (16HBE). B, SPC-A1, H1299, and PC9 cells were transfected with si-ANRIL. C, MTTassays were used to determine the cell viability for si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. Values represent the mean � SD fromthree independent experiments. D, colony-forming assays were conducted to determine the proliferation of si-ANRIL–transfected SPC-A1, H1299, and PC9cells. Flow-cytometry assays were performed to analysis the cell-cycle progression and apoptosis when NSCLC cells transfected with si-ANRIL; � , P < 0.05 and�� , P < 0.01.






investigate the correlation between ANRIL and KLF2 expressions.Two-sided P values were calculated, and a probability level of0.05 was chosen for statistical significance.

ResultsANRIL expression was upregulated and correlated with poorprognosis of NSCLC

ANRIL expression levels were investigated in 68 pairedNSCLC samples and adjacent histologically normal tissuesusing qPCR assays. ANRIL expression was significantly upre-gulated (fold change >1.5, P < 0.01) in 76% (52/68) ofcancerous tissues compared with normal tissues (Fig. 1A);the ANRIL expression level in each patient was shown inSupplementary Table S2. Increased ANRIL expression levelsin NSCLC were significantly correlated with tumor size (P ¼0.001), and advanced pathologic stage (P ¼ 0.007). However,ANRIL expression was not associated with other parameterssuch as gender (P ¼ 0.625) and age (P ¼ 0.627) in NSCLC(Table 1).

To investigate whether upregulation of ANRIL is caused byDNA copy-number variation, we referred to the array comparativegenomic hybridization (aCGH) database in GSE20393, wheredeposits of 52 lung cancer copy-number alteration data weregenerated by Agilent Human Genome CGH 244A Microarrays.We investigated 26 probes representing region of ANRIL andextracted GLAD-segmented copy number of these probes. Theresults showed that there is no significant gain of DNA copynumber in this region, suggesting that upregulation of ANRIL inlung cancer is not due to copy-number variation (SupplementaryFig. S1A).

Association of ANRIL expression with patients' survivalKaplan–Meier survival analysis was conducted to investigate

the correlation between ANRIL expression and NSCLC patientprognosis. According to relative ANRIL expression in tumortissues, the 68 patients with NSCLC were classified into twogroups: the high ANRIL group (n ¼ 34, fold-change � meanratio); and the low ANRIL group (n ¼ 34, fold-change � meanratio; Fig. 1B).With respect to progression-free survival (PFS), thiswas 35.3% for the low ANRIL group, and 13.6% for the highANRIL group. Median survival time for the low ANRIL group was31 months, and 14 months for the high ANRIL group (Fig. 1C).The OS rate over 3 years for the low ANRIL group was 44.4%, and20.8% for the highANRIL group.Median survival time for the lowANRIL group was 32 months, and 18 months for the high ANRILgroup (Fig. 1D).

Univariate analysis identified three prognostic factors: lymphnode metastasis; TNM stage; and ANRIL expression level. Otherclinicopathologic features such as gender and age were not sta-tistically significant prognosis factors (Supplementary Table S3).Multivariate analysis of the three prognosis factors confirmed thatHR for ARAIL expression is 3.509 (95% confidence interval,1.619–7.607) of PFS, indicating that ANRIL expressionmay serveas a potential independent prognostic value in NSCLC (Supple-mentary Table S4).

Modulation of ANRIL expression in NSCLC cellsWe next performed qPCR analysis to examine the expression

of ANRIL in 6 human NSCLC cell lines, including bothadenocarcinoma and squamous carcinoma subtypes (Fig.2A). To investigate the functional effects of ANRIL in NSCLCcells, we modulated its expression through RNAi. qPCR

Figure 3.Effects of knockdown of ANRIL onNSCLC cell cycle and apoptosis in vitro. A, the bar chart represents the percentage of cells in G0–G1, S, or G2–Mphase, as indicated.B, apoptosis was determined by flow cytometry; upper left, necrotic cells; upper right, terminal apoptotic cells; lower right, early apoptotic cells. C, apoptosis wasdetermined by Tunel staining. All experiments were performed in biologic triplicates with three technical replicates; �� , P < 0.01.

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analysis of ANRIL levels was performed 48 hours after trans-fection. ANRIL expression was knocked down by 74% inSPC-A1 cells, 75% in H1299 cells, and 94% in PC9 cellsby si-ANRIL transfection when compared with control cells(si-NC; Fig. 2B).

Knockdown of ANRIL impaired NSCLC cells proliferation andinduced apoptosis

To assess the role of ANRIL inNSCLC, we investigated the effectof targeted knockdown of ANRIL on cell proliferation.MTT assaysrevealed that cell growth was inhibited in SPC-A1, H1299, andPC9 cells transiently transfected with si-ANRIL compared withcontrols (Fig. 2C). Meanwhile, knockdown of ANRIL expressioncould also inhibit A549 cells (with relative low endogenousANRIL expression level) proliferation (Supplementary Fig.S1B). Colony formation assay results revealed that clonogenicsurvival was inhibited following downregulation of ANRIL inSPC-A1,H1299, andPC9cells (Fig. 2D).However, overexpressionof ANRIL in 16HBE cells showed no significant effect on cellproliferation (Supplementary Fig. S1C).

To further examine whether the effect of knockdown ANRIL onproliferation of NSCLC cells reflected cell-cycle arrest, cell-cycleprogression was analyzed by flow-cytometry analysis. The resultsrevealed that SPC-A1 and PC9 cells transfected with si-ANRIL hadan obvious cell-cycle arrest at the G1–G0 phase and had adecreased G2–S phase (Fig. 3A). To determine whether NSCLCcell proliferation was influenced by cell apoptosis, we performedflow-cytometry and Tunel staining analysis. The results showed

that NSCLC cells transfected with ANRIL siRNA promoted apo-ptosis in comparison with that in control cells (Fig. 3B and 2C).These data indicate that ANRIL could promote the proliferationphenotype of NSCLC cells.

Decreased ANRIL expression inhibits NSCLC cells migrationTo investigate the effect of ANRIL knockdown on NSCLC cells

migration, Transwells assays were performed. The results showedthat decreased ANRIL expression levels impeded the migration ofSPC-A1 and PC9 cells compared with controls (SupplementaryFig. S1D).

Downregulation of ANRIL inhibits NSCLC cells tumorigenesisin vivo

To explore whether the level of ANRIL expression could affecttumorigenesis, PC9 cells stably transfected with sh-ANRIL orempty vector were inoculated into female nude mice. Eighteendays after the injection, the tumors formed in the sh-ANRILgroup were substantially smaller than those in the control group(Fig. 4A). Moreover, the mean tumor weight at the end of theexperiment was markedly lower in the sh-ANRIL group (0.62 �0.35 g) comparedwith the empty vector group (1.41� 0.57 g; Fig.4B). qPCR analysis found that the levels of ANRIL expression intumor tissues formed from sh-ANRIL cells were lower than intumors formed in the control group (Fig. 4C). Tumors formedfrom sh-ANRIL–transfected PC9 cells exhibited decreased positivefor Ki67 than those from control cells (Fig. 4D). These findingsindicate that knockdown of ANRIL inhibits tumor growth in vivo.

Figure 4.Effects of downregulation of ANRIL on tumor growth in vivo. A, the tumor volume was calculated once every 3 days after injection of PC9 cells stably transfectedwith sh-ANRIL or empty vector. Points, mean (n ¼ 7); bars, SD. B, tumor weights are represented as means of tumor weights � SD. C, qPCR analysis ofANRIL expression in tumor tissues formed from PC9/sh-ANRIL, PC9/empty vector. D, tumors developed from sh-ANRIL–transfected PC9 cells showed lower Ki67protein levels than tumors developed by control cells. Top, H&E staining; bottom, immunostaining; � , P < 0.05 and �� , P < 0.01.






ANRIL silences KLF2 and P21 transcription by binding withEZH2

Previously studies have indicated that ANRIL could silencep15INK4 transcription and contribute to cancer cells proliferation(18). The results of qPCR showed that p15 and p16 expressionwas increased in SPCA1 and H1299 cells with transfection of si-ANRIL; however, there was no significant difference of p15expression in PC9 cells when knockdown of ANRIL expression(Fig. 5A and Supplementary Fig. S1E). There are evidence thatshowed that EZH2 could regulate KLF2 and P21 expression (20,21), and our qPCR results also showed that inhibition of ANRILexpression led to increased KLF2 and P21 expression. Moreover,knockdown of EZH2 or SUZ12 could also upregulate KLF2 andP21 expression (Fig. 5B). Meanwhile, the Western blot assaysshowed the same results (Fig. 5C), which indicated that KLF2and P21 could be ANRIL novel targets in PC9 cells. In addition,we found that ANRIL RNAs were mostly located in the nucleus(Fig. 5D).

To further investigate whether ANRIL repress KLF2 and P21expression through binding PRC2,we performedRIP analysis andthe results showed that ANRIL could directly bind with EZH2 inPC9 cells (Fig. 6A). Furthermore, the results of ChIP assaysshowed that EZH2 could directly bind to KLF2 and P21 promoterregion andmediate H3K27me3modification (Fig. 6B). However,knockdown of ANRIL reduced EZH2 binding with KLF2 and P21promoter (Fig. 6C). Finally, we detected the KLF2 expression inNSCLC tissues, and found that there is an inverse relationshipbetween ANRIL and KLF2 expression (Fig. 6D). These data sug-gested that ANRIL promotes NSCLC PC9 cells proliferation is notdependent on regulation p15 expression, but also through silenc-ing of KLF2 and P21 transcription.

Silence of KLF2 is potentially involved in the oncogenicfunction of ANRIL

To investigate whether KLF2 is involved in the ANRIL-inducedincrease in NSCLC cell proliferation, we performed gain-of-func-tion assays. The results ofWestern blot analysis showed that KLF2expression was significantly upregulated in PC9 cells transfectedwith pCDNA–KLF2 compared with control cells (Fig. 7A). Mean-while, MTT and colony formation assay results revealed thatoverexpression of KLF2 could impaired NSCLC cells proliferation(Fig. 7B). Moreover, flow-cytometry analysis indicated thatincreased KLF2 expression could induce NSCLC cells apoptosis(Fig. 7C). Furthermore, to determine whether ANRIL regulateNSCLC cell proliferation via repressing KLF2 expression, rescueassays were performed. PC9 cells were cotransfected with si-ANRIL and si-KLF2, and this was shown to rescue the decreasedexpression of ANRIL induced by knockdown of KLF2 (Fig. 7D).The results of MTT and colony formation assay results indicatedthat cotransfection could partially rescue si-ANRIL–impairedproliferation in PC9 cells (Fig. 7E). These data indicate thatANRIL promotes NSCLC cell proliferation through the down-regulation of KLF2 expression.

DiscussionRecently, numerous pieces of evidence show that many

lncRNAs are characterized and play important roles in cancerpathogenesis, suggesting that they could provide new insightsinto the biology of this disease. For example, increased lncRNAHOTTIP is associated with progression and predicts outcomein patients with hepatocellular carcinoma by regulatingHOXA13 expression (22). However, the roles of lncRNAs in

Figure 5.ANRIL could silenceKLF2 andP21 expression. A, the levels of p15INK4B andp16mRNAweredeterminedbyqPCR in SPC-A1 andPC9 cells transfectedwith si-ANRIL andresults are expressed relative to the corresponding values for control cells. B and C, the levels of p21 and KLF2 mRNA and protein levels were determined byqPCR andWestern blotwhen PC9 cells were transfectedwith si-EZH2 or si-SUZ12. D, ANRIL expression levels in cell cytoplasmor nucleus of NSCLC cell lines SPC-A1,PC9, and H520 were detected by qPCR. � , P < 0.05 and �� , P < 0.01; N.S., not significant.

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NSCLC are still not well documented, and one of theselncRNAs is metastasis-associated lung adenocarcinoma tran-script 1 (MALAT1), which is a highly conserved nuclear lncRNAand a predictive marker for metastasis development in lungcancer (23). In our previous studies, we found that increasedlncRNA HOTAIR–promoted NSCLC cells invasion and metas-tasis via regulating HOXA5 expression, and lncRNA BANCRoverexpression could impaired NSCLC cells proliferationand metastasis by affecting epithelial–mesenchymal transition(24, 25).

In this study, we demonstrated that the expression of anotherlncRNA, ANRIL, is significantly upregulated in NSCLC tissues.Specifically, increased ANRIL expression appears to be a signifi-cant, independent predictive value for patients with NSCLC.Moreover, knockdown of ANRIL expression led to the significantinhibition of cell proliferation and the promotion of apoptosisboth in vitro and in vivo. These findings suggest that ANRIL plays adirect role in the modulation of cell proliferation and NSCLCprogression, and could be a useful novel prognostic or progres-sion marker for NSCLC. As more and more lncRNAs are studied,many have been shown to function by binding with PRC2 andsilencing downstream target genes that involved in multiplecancers, including NSCLC (26, 27). ANRIL has been reported toinvolve in cancer cells proliferation by silencing p15INK4 expres-sion. In this study, we found that ANRIL is mostly located in cellnucleus and could directly bind with EZH2, a core subunit ofPRC2, resulted in repressing KLF2 and P21 transcription. How-ever, knockdown of ANRIL could not influence p15INK4 expres-

sion in NSCLC PC9 cells, which indicated that ANRIL contributedto NSCLC cell proliferation is not dependent on regulatingp15INK4, but also could through silencing KLF2 and P21transcription.

The Kruppel-like factor (KLF) family transcription factors,with Cys2/His2 zinc-finger domains, could function as sup-pressors or activators in a cell type and promoter-dependentmanner and involve in cell differentiation and proliferation(28, 29). Some KLF members are emerging as tumor-suppressorgenes due to their roles in the inhibition of proliferation,migration, and induction of apoptosis (30, 31). KLF2, as amember of KLF family, is diminished in many cancers andpossesses tumor-suppressor features such as inhibition of cellproliferation mediated by KRAS (32–34). Moreover, there isevidence that showed that EZH2 could silence KLF2 expressionand block the tumor-suppressor features of KLF2, which ispartly mediated by p21 (21). Our results also showed thatlncRNA ANRIL takes part in NSCLC cells' proliferation byepigenetic-silencing KLF2 and P21 transcription, and KLF2inactivation further led to the decreased P21 expression. Asmore and more studies indicated that lncRNAs are oftenexpressed in a spatial- or temporal-specific pattern, and morecell- and tissue-specific pattern. Our results also showed thateven in NSCLC cells, lncRNA ANRIL could regulate differenttarget genes in different cell lines, which suggested that lncRNA,especially ANRIL, can influence the same cell biologic functionvia regulating different target genes dependent on different celllines.

Figure 6.ANRIL could directly bind PRC2 and silence KLF2 and P21 transcription. A, RIPwith rabbitmonoclonal anti-EZH2, preimmune IgG, or 10% input from PC9 cell extracts.RNA levels in immunoprecipitates were determined by qPCR. Expression levels of ANRIL RNA are presented as fold enrichment in EZH2 relative to IgGimmunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates were used as positive control. B and C, ChIP–qPCR ofEZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells, and IgG as a negative control; ChIP–qPCR of EZH2 occupancy and H3K27-3mebinding in the KLF2 promoter in PC9 cells treated with ANRIL siRNA (48 hours) or scrambled siRNA. D, analysis of the relationship between ANRIL expression andKLF2 mRNA level (DCt value) in 40 NSCLC tissues. The mean values and SDs were calculated from triplicates of a representative experiment.






To date, although only a small number of lncRNAs have beenwell characterized, they have been shown to regulate gene expres-sion at various levels, including chromatin modification and post-transcriptional processing (35, 36). Here, the possible other targetsand mechanisms that underlie such regulatory behaviors stillremain to be fully understood despite our observation ofANRIL-induced NSCLC cell proliferation. In summary, the expres-sion of ANRIL was significantly increased in NSCLC tissues, sug-gesting that its upregulationmay be a negative prognostic factor forpatients withNSCLC, indicative of poor survival rates, and a higherrisk for cancermetastasis.We showed thatANRILpossibly regulatesthe proliferation ability of NSCLC cells, partially through its reg-ulation of the KLF2 and P21, which indicated that lncRNAs con-tribute to different cancer cells biologic function maybe throughregulating different target genes. Our findings further the under-standing ofNSCLCpathogenesis, and facilitate the development oflncRNA-directed diagnostics and therapeutics against cancers.

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

Authors' ContributionsConception and design: F.-q. Nie, M. Sun, T.-p. Xu, K.-h. Lu, Y.-q. ShuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-s. Yang, R. Xia, X.-h. Liu, E.-b. Zhang

Analysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): F.-q. Nie, J.-s. Yang, M. Xie, R. Xia, Y.-w. Liu,X.-h. LiuWriting, review, and/or revision of the manuscript: F.-q. Nie, K.-h. Lu,Y.-q. ShuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.-b. ZhangStudy supervision: M. Sun, K.-h. Lu, Y.-q. Shu

AcknowledgmentsThe authors are very grateful to professor Xiongbin Lu for providing the

ANRIL overexpression plasmid.

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

Foundation of China (81372397; to K.-h. Lu); (81301824; to X.-h. Liu);(81172140 and81272532; toY.-q. Shu). Thisworkwas also supportedbyPriorityAcademic Program Development of Jiangsu Higher Education Institutions(JX10231801). M. Sun was supported by a Jiangsu province ordinary universitygraduate student research innovation project for 2013 (CXZZ13_0562,JX22013265).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 13, 2014; revised October 21, 2014; accepted November 7,2014; published OnlineFirst December 12, 2014.

Figure 7.Overexpression of KLF2 expression inhibits PC9 cells proliferation. PC9 cells were transfected with pCDNA–KLF2 or cotransfected with si-ANRIL and si-KLF2. A, theprotein level of KLF2 in PC9 cells transfected with pCDNA–KLF2 was detected by Western blot analysis. B, MTT assays and colony-forming assays were usedto determine the cell viability for pCDNA–KLF2-transfected PC9 cells. Values represent the mean � SD from three independent experiments. C, apoptosiswas determined by flow cytometry. Upper left, necrotic cells; upper right, terminal apoptotic cells; lower right, early apoptotic cells. D, the protein level of KLF2 in PC9cells cotransfected with si-ANRIL and si-KLF2 was detected by Western blot analysis. E, MTT assays and colony-forming assays were used to determine the cellviability for si-ANRIL and si-KLF2 cotransfected PC9 cells. Values represent the mean � SD from three independent experiments; � , P < 0.05 and �� , P < 0.01.


Nie et al.




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2015;14:268-277. Published OnlineFirst December 12, 2014.Mol Cancer Ther Feng-qi Nie, Ming Sun, Jin-song Yang, et al. and P21 ExpressionCancer Cell Proliferation and Inhibits Apoptosis by Silencing KLF2

Small Cell Lung−Long Noncoding RNA ANRIL Promotes Non

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