Recent evidence highlights long noncoding RNAs (lncRNA) as crucial regulators of cancer biology that contribute to essential cancer cell functions such as cell proliferation, apoptosis, and metastasis. In non–small cell lung cancer (NSCLC), several lncRNAs' expressions are misregulated and have been nominated as critical actors in NSCLC tumorigenesis. LncRNA ANRIL was first found to be required for the PRC2 recruitment to and silencing of p15INK4B, the expression of which is induced by the ATM–E2F1 signaling pathway. Our previous study showed that ANRIL was significantly upregulated in gastric cancer, and it could promote cell proliferation and inhibit cell apoptosis by silencing of miR99a and miR449a transcription. However, its clinical significance and potential role in NSCLC is still not documented. In this study, we reported that ANRIL expression was increased in NSCLC tissues, and its expression level was significantly correlated with tumor–node–metastasis stages and tumor size. Moreover, patients with high levels of ANRIL expression had a relatively poor prognosis. In addition, taking advantage of loss-of-function experiments in NSCLC cells, we found that knockdown of ANRIL expression could impair cell proliferation and induce cell apoptosis both in vitro and vivo. Furthermore, we uncover that ANRIL could not repress p15 expression in PC9 cells, but through silencing of KLF2 and P21 transcription. Thus, we conclusively demonstrate that lncRNA ANRIL plays a key role in NSCLC development by associating its expression with survival in patients with NSCLC, providing novel insights on the function of lncRNA-driven tumorigenesis. Mol Cancer Ther; 14(1); 268–77. ©2014 AACR.

Lung 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 lung cancer cases, represents the most prevalent class of this cancer type, and includes several histologic subtypes such as squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma (LCC; refs. 2, 3). Despite current advances in the treatments for NSCLC, including surgical therapy, chemotherapy, and molecular targeting therapy, the overall 5-year survival rate for patients with NSCLC has not been markedly improved 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 is essential for the developing of specific diagnostic methods and designing of more individualized and effective therapeutic strategies.

Recently, studies using the great advances in genomic technologies have revealed the majority of the human genome is transcribed, whereas only 2% of the transcribed genome codes for protein (5). Meanwhile, it is becoming increasingly apparent that the large majority of genome is transcribed into noncoding RNAs (ncRNAs), including microRNAs and long ncRNAs (lncRNAs; ref. 6). The ENCODE Consortium has elucidated the prevalence of thousands of human lncRNAs, but only very few of them have been assigned with any biologic function (7). To date, studies showed that miRNAs play important roles in the posttranscriptional regulation of gene expression; however, the lncRNAs counterpart of ncRNA is not well characterized (8). Although very few are characterized in detail, LncRNAs are involved in a large range of biologic processes, including modulation of apoptosis and invasion, reprogramming stem cell pluripotency, and parental imprinting through the regulation of gene expression by chromatin remodeling, histone protein modification, regulation of mRNA splicing, and acting as sponges for microRNAs (9–12).

In the past decade, lots of evidence have linked the dysregulation of lncRNAs with diverse human diseases, in particular cancers (13–15). Therefore, identification of cancer-associated lncRNAs and investigation of their molecular and biologic functions are important in understanding the molecular biology of NSCLC development and progression. Our previous study showed that lncRNA ANRIL was significantly upregulated in gastric cancer, and increased ANRIL promoted gastric cancer cells proliferation and inhibited apoptosis by epigenetic silencing of miR99a and miR449a transcription (16). Moreover, ANRIL can bind to 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 NSCLC development and progression is still not documented.

In this study, we found that lncRNA ANRIL expression was increased in NSCLC tissues compared with adjacent normal tissues. Its expression level was significantly correlated with tumor–node–metastasis (TNM) stages and tumor size. Moreover, patients with higher level of ANRIL expression had a relatively poor prognosis. Furthermore, we investigated the effects of ANRIL expression on NSCLC cell phenotype in vitro and in vivo with the loss-of-function study. Moreover, we also showed that ANRIL could bind to PRC2 to repress KLF2 and P21 transcription, but not regulate P15INK4 expression in NSCLC PC9 cells, which indicated that ANRIL affected NSCLC cells proliferation and apoptosis partly via silencing of KLF2 and P21 transcription. This study advances our understanding of the role of lncRNAs, such as a regulator of pathogenesis of NSCLC and facilitates the development of lncRNA-directed diagnostics and therapeutics.

Tissue collection

We obtained 68 paired NSCLC and adjacent nontumor lung tissues from patients who underwent surgery at Jiangsu Province Hospital between 2010 and 2011, and were diagnosed with NSCLC based on histopathologic evaluation. Clinicopathologic characteristics, including TNM staging, were recorded. No local or systemic treatment was conducted in these patients before surgery. All collected tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until required. Our study was approved by the Research Ethics Committee of Nanjing Medical University, China. Written informed consent was obtained from all patients.

Cell lines

Five NSCLC adenocarcinoma cell lines (PC9, SPC-A1, NCI-H1975, H1299, and H358), and one NSCLC squamous carcinomas cell lines (H520) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). A549, H1975, H1299, and H520 cells were cultured in RPMI-1640; 16HBE, PC9, and SPC-A1 cells were cultured in DMEM (GIBCO-BRL) medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad) at 37ºC/5% CO2. All cell lines were authenticated by short tandem repeat DNA profiling.

RNA extraction and qPCR assays

Total RNA was isolated with TRizol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (500 ng) was reverse transcribed in a final volume of 10 μL using random primers under standard conditions for the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). We used the SYBR Premix Ex Taq (TaKaRa, Dalian, China) to determine ANRIL expression levels, following the manufacturer's instructions. Results were normalized to the expression of GAPDH. The specific primers used are shown in Supplementary Table S1. The qPCR assays were conducted on an ABI 7500, and data collected with this instrument. Our qPCR results were analyzed and expressed relative to threshold cycle (Ct) values, and then converted to fold changes.

Cell transfection

Human ANRIL cDNA clone L6ChoCKO-2-E10 with functional region was provided by the Functional Genomics Research Center, KRIBB, Korea. Plasmid vectors (pCNS-ANRIL, sh-ANRIL and empty vector) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen), and transfected into16HBE, SPC-A1, H1299, or PC9cells. The si-ANRIL or si-NC was transfected into SPC-A1, H1299, or PC9 cells. SPC-A1, H1299, or PC9 cells were grown on 6-well plates to confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 48 hours after transfection, cells were harvested for qPCR or Western blot analysis.

Cell viability assays

Cell viability was monitored using a Cell Proliferation Reagent Kit I (MTT; Roche Applied Science). The SPC-A1, H1299, PC9, or A549 cells transfected with si-ANRIL (3000 cells/well) were grown in 96-well plates. Cell viability was assessed every 24 hours following the manufacturer's protocol. All experiments were performed in quadruplicate. For each treatment group wells were assessed in triplicate.

Flow cytometry

SPC-A1, H1299, or PC9 cells transfected with si-ANRIL were harvested 48 hours after transfection by trypsinization. After the double staining with FITC–Annexin V and Propidium iodide (PI) was done using the FITC–Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's recommendations, the cells were analyzed with a flow cytometry (FACScan; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells, and then the relative ratio of early apoptotic cells were compared with control transfectant from each experiment. Cells for cell–cycle analysis were stained with PI using the CycleTEST PLUS DNA Reagent Kit (BD Biosciences) following the protocol and analyzed by FACScan. The percentage of the cells in G0–G1, S, and G2–M phase were counted and compared.

Tumor formation assay in a nude mouse model

Female athymic BALB/c nude mice (4-weeks-old) were maintained under pathogen-free conditions and manipulated according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. PC9 cells were stably transfected with sh-ANRIL and empty vector and harvested from 6-well cell culture plates, washed with PBS, and resuspended at a concentration of 1 × 108 cells/mL. A total of 100 μL of suspended cells was s.c. injected into a single side of the posterior flank of each mouse. Tumor growth was examined every 3 days, and tumor volumes were calculated using the equation V = 0.5 × D × d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). At 18 days after injection, mice were euthanized, and the subcutaneous growth of each tumor was examined. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Nanjing medical University.

RNA immunoprecipitation

For immunoprecipitation (IP) of endogenous PRC2 complexes from whole-cell extracts, cells were lysed. The supernatants were incubated with protein A Sepharose beads coated with antibodies that recognized EZH2, SNRNP70, or with control IgG (Millipore) 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 Proteinase K (30 minutes at 55°C) to remove proteins, respectively. The PRC2 isolated from the IP materials was further assessed by qPCR analysis (19).

Chromatin immunoprecipitation

PC9 cells were treated with formaldehyde and incubated for 10 minutes to generate DNA–protein cross-links. Cell lysates were then sonicated to generate chromatin fragments of 200 to 300 bp and immunoprecipitated with EZH2 and H3K27me3-specific antibody (Cell Signaling Technology) or IgG as control. Precipitated chromatin DNA was recovered and analyzed by qPCR.

Western blot assay and antibodies

Cells protein lysates were separated by 10% SDSPAGE, transferred to 0.22 μm NC membranes (Sigma), and incubated with specific antibodies. ECL chromogenic substrate was used and 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 from Cell Signaling Technology, Inc.; anti-KLF2 was purchased from Sigma.

Statistical analysis

All statistical analyses were performed using SPSS 17.0 software (IBM). The significance of differences between groups was estimated by the Student t test, Wilcoxon test, or χ2 test. Disease-free survival (DFS) and overall survival (OS) rates were calculated by the Kaplan–Meier method with the log-rank test applied for comparison. The date of survival was evaluated by univariate and multivariate Cox proportional hazards models. Variables with P < 0.05 in univariate analysis were used in subsequent multivariate analysis on the basis of Cox regression analyses. Kendall Tau-b and Pearson correlation analyses were used to investigate the correlation between ANRIL and KLF2 expressions. Two-sided P values were calculated, and a probability level of 0.05 was chosen for statistical significance.

ANRIL expression was upregulated and correlated with poor prognosis of NSCLC

ANRIL expression levels were investigated in 68 paired NSCLC samples and adjacent histologically normal tissues using qPCR assays. ANRIL expression was significantly upregulated (fold change >1.5, P < 0.01) in 76% (52/68) of cancerous tissues compared with normal tissues (Fig. 1A); the ANRIL expression level in each patient was shown in Supplementary Table S2. Increased ANRIL expression levels in NSCLC were significantly correlated with tumor size (P = 0.001), and advanced pathologic stage (P = 0.007). However, ANRIL expression was not associated with other parameters such as gender (P = 0.625) and age (P = 0.627) in NSCLC (Table 1).

Figure 1.

Relative ANRIL expression in NSCLC tissues and its clinical significance. A, relative expression of ANRIL in NSCLC tissues (n = 68) compared with corresponding nontumor tissues (n = 68). ANRIL expression was examined by qPCR and normalized to GAPDH expression. Results are presented as the fold change in 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 ANRIL expression levels.

Figure 1.

Relative ANRIL expression in NSCLC tissues and its clinical significance. A, relative expression of ANRIL in NSCLC tissues (n = 68) compared with corresponding nontumor tissues (n = 68). ANRIL expression was examined by qPCR and normalized to GAPDH expression. Results are presented as the fold change in 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 ANRIL expression levels.

Close modal
Table 1.

Correlation between ANRIL expression and clinicopathologic characteristics of NSCLC patients

ANRIL
CharacteristicsHigh, number of cases (34)Low, number of cases (34)χ2 test (P value)
Age, y   0.627 
 ≤65 17 19  
 >65 17 15  
Gender   0.625 
 Male 18 20  
 Female 16 14  
Histologic subtype   0.324 
 SCC 22 18  
 Adenocarcinoma 12 16  
TNM stage   0.007a 
 Ia + Ib 15  
 IIa + IIb 14 12  
 IIIa 16  
Tumor size   0.001a 
 ≤5 cm 13 26  
 >5 cm 21  
Lymph node metastasis   0.051 
 Negative 11 19  
 Positive 23 15  
Smoking history   0.793 
 Smokers 23 24  
 Never smokers 11 10  
ANRIL
CharacteristicsHigh, number of cases (34)Low, number of cases (34)χ2 test (P value)
Age, y   0.627 
 ≤65 17 19  
 >65 17 15  
Gender   0.625 
 Male 18 20  
 Female 16 14  
Histologic subtype   0.324 
 SCC 22 18  
 Adenocarcinoma 12 16  
TNM stage   0.007a 
 Ia + Ib 15  
 IIa + IIb 14 12  
 IIIa 16  
Tumor size   0.001a 
 ≤5 cm 13 26  
 >5 cm 21  
Lymph node metastasis   0.051 
 Negative 11 19  
 Positive 23 15  
Smoking history   0.793 
 Smokers 23 24  
 Never smokers 11 10  

aOverall P < 0.05.

To investigate whether upregulation of ANRIL is caused by DNA copy-number variation, we referred to the array comparative genomic hybridization (aCGH) database in GSE20393, where deposits of 52 lung cancer copy-number alteration data were generated by Agilent Human Genome CGH 244A Microarrays. We investigated 26 probes representing region of ANRIL and extracted GLAD-segmented copy number of these probes. The results showed that there is no significant gain of DNA copy number in this region, suggesting that upregulation of ANRIL in lung cancer is not due to copy-number variation (Supplementary Fig. S1A).

Association of ANRIL expression with patients' survival

Kaplan–Meier survival analysis was conducted to investigate the correlation between ANRIL expression and NSCLC patient prognosis. According to relative ANRIL expression in tumor tissues, the 68 patients with NSCLC were classified into two groups: the high ANRIL group (n = 34, fold-change ≤ mean ratio); and the low ANRIL group (n = 34, fold-change ≥ mean ratio; Fig. 1B). With respect to progression-free survival (PFS), this was 35.3% for the low ANRIL group, and 13.6% for the high ANRIL group. Median survival time for the low ANRIL group was 31 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%, and 20.8% for the high ANRIL group. Median survival time for the low ANRIL group was 32 months, and 18 months for the high ANRIL group (Fig. 1D).

Univariate analysis identified three prognostic factors: lymph node metastasis; TNM stage; and ANRIL expression level. Other clinicopathologic features such as gender and age were not statistically significant prognosis factors (Supplementary Table S3). Multivariate analysis of the three prognosis factors confirmed that HR for ARAIL expression is 3.509 (95% confidence interval, 1.619–7.607) of PFS, indicating that ANRIL expression may serve as a potential independent prognostic value in NSCLC (Supplementary Table S4).

Modulation of ANRIL expression in NSCLC cells

We next performed qPCR analysis to examine the expression of ANRIL in 6 human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes (Fig. 2A). To investigate the functional effects of ANRIL in NSCLC cells, we modulated its expression through RNAi. qPCR analysis of ANRIL levels was performed 48 hours after transfection. ANRIL expression was knocked down by 74% in SPC-A1 cells, 75% in H1299 cells, and 94% in PC9 cells by si-ANRIL transfection when compared with control cells (si-NC; Fig. 2B).

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 and H520) compared with that in normal human bronchial epithelial cells (16HBE). B, SPC-A1, H1299, and PC9 cells were transfected with si-ANRIL. C, MTT assays were used to determine the cell viability for si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. Values represent the mean ± SD from three independent experiments. D, colony-forming assays were conducted to determine the proliferation of si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. 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.

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 and H520) compared with that in normal human bronchial epithelial cells (16HBE). B, SPC-A1, H1299, and PC9 cells were transfected with si-ANRIL. C, MTT assays were used to determine the cell viability for si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. Values represent the mean ± SD from three independent experiments. D, colony-forming assays were conducted to determine the proliferation of si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. 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.

Close modal

Knockdown of ANRIL impaired NSCLC cells proliferation and induced apoptosis

To assess the role of ANRIL in NSCLC, we investigated the effect of targeted knockdown of ANRIL on cell proliferation. MTT assays revealed that cell growth was inhibited in SPC-A1, H1299, and PC9 cells transiently transfected with si-ANRIL compared with controls (Fig. 2C). Meanwhile, knockdown of ANRIL expression could also inhibit A549 cells (with relative low endogenous ANRIL expression level) proliferation (Supplementary Fig. S1B). Colony formation assay results revealed that clonogenic survival was inhibited following downregulation of ANRIL in SPC-A1, H1299, and PC9cells (Fig. 2D). However, overexpression of ANRIL in 16HBE cells showed no significant effect on cell proliferation (Supplementary Fig. S1C).

To further examine whether the effect of knockdown ANRIL on proliferation of NSCLC cells reflected cell-cycle arrest, cell-cycle progression was analyzed by flow-cytometry analysis. The results revealed that SPC-A1 and PC9 cells transfected with si-ANRIL had an obvious cell-cycle arrest at the G1–G0 phase and had a decreased G2–S phase (Fig. 3A). To determine whether NSCLC cell proliferation was influenced by cell apoptosis, we performed flow-cytometry and Tunel staining analysis. The results showed that NSCLC cells transfected with ANRIL siRNA promoted apoptosis in comparison with that in control cells (Fig. 3B and 2C). These data indicate that ANRIL could promote the proliferation phenotype of NSCLC cells.

Figure 3.

Effects of knockdown of ANRIL on NSCLC cell cycle and apoptosis in vitro. A, the bar chart represents the percentage of cells in G0–G1, S, or G2–M phase, 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 was determined by Tunel staining. All experiments were performed in biologic triplicates with three technical replicates; **, P < 0.01.

Figure 3.

Effects of knockdown of ANRIL on NSCLC cell cycle and apoptosis in vitro. A, the bar chart represents the percentage of cells in G0–G1, S, or G2–M phase, 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 was determined by Tunel staining. All experiments were performed in biologic triplicates with three technical replicates; **, P < 0.01.

Close modal

Decreased ANRIL expression inhibits NSCLC cells migration

To investigate the effect of ANRIL knockdown on NSCLC cells migration, Transwells assays were performed. The results showed that decreased ANRIL expression levels impeded the migration of SPC-A1 and PC9 cells compared with controls (Supplementary Fig. S1D).

Downregulation of ANRIL inhibits NSCLC cells tumorigenesis in vivo

To explore whether the level of ANRIL expression could affect tumorigenesis, PC9 cells stably transfected with sh-ANRIL or empty vector were inoculated into female nude mice. Eighteen days after the injection, the tumors formed in the sh-ANRIL group were substantially smaller than those in the control group (Fig. 4A). Moreover, the mean tumor weight at the end of the experiment was markedly lower in the sh-ANRIL group (0.62 ± 0.35 g) compared with the empty vector group (1.41 ± 0.57 g; Fig. 4B). qPCR analysis found that the levels of ANRIL expression in tumor tissues formed from sh-ANRIL cells were lower than in tumors formed in the control group (Fig. 4C). Tumors formed from sh-ANRIL–transfected PC9 cells exhibited decreased positive for Ki67 than those from control cells (Fig. 4D). These findings indicate 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 transfected with sh-ANRIL or empty vector. Points, mean (n = 7); bars, SD. B, tumor weights are represented as means of tumor weights ± SD. C, qPCR analysis of ANRIL expression in tumor tissues formed from PC9/sh-ANRIL, PC9/empty vector. D, tumors developed from sh-ANRIL–transfected PC9 cells showed lower Ki67 protein levels than tumors developed by control cells. Top, H&E staining; bottom, immunostaining; *, P < 0.05 and **, P < 0.01.

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 transfected with sh-ANRIL or empty vector. Points, mean (n = 7); bars, SD. B, tumor weights are represented as means of tumor weights ± SD. C, qPCR analysis of ANRIL expression in tumor tissues formed from PC9/sh-ANRIL, PC9/empty vector. D, tumors developed from sh-ANRIL–transfected PC9 cells showed lower Ki67 protein levels than tumors developed by control cells. Top, H&E staining; bottom, immunostaining; *, P < 0.05 and **, P < 0.01.

Close modal

ANRIL silences KLF2 and P21 transcription by binding with EZH2

Previously studies have indicated that ANRIL could silence p15INK4 transcription and contribute to cancer cells proliferation (18). The results of qPCR showed that p15 and p16 expression was increased in SPCA1 and H1299 cells with transfection of si-ANRIL; however, there was no significant difference of p15 expression in PC9 cells when knockdown of ANRIL expression (Fig. 5A and Supplementary Fig. S1E). There are evidence that showed that EZH2 could regulate KLF2 and P21 expression (20, 21), and our qPCR results also showed that inhibition of ANRIL expression led to increased KLF2 and P21 expression. Moreover, knockdown of EZH2 or SUZ12 could also upregulate KLF2 and P21 expression (Fig. 5B). Meanwhile, the Western blot assays showed the same results (Fig. 5C), which indicated that KLF2 and P21 could be ANRIL novel targets in PC9 cells. In addition, we found that ANRIL RNAs were mostly located in the nucleus (Fig. 5D).

Figure 5.

ANRIL could silence KLF2 and P21 expression. A, the levels of p15INK4B and p16 mRNA were determined by qPCR in SPC-A1 and PC9 cells transfected with si-ANRIL and results 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 by qPCR and Western blot when PC9 cells were transfected with si-EZH2 or si-SUZ12. D, ANRIL expression levels in cell cytoplasm or 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.

Figure 5.

ANRIL could silence KLF2 and P21 expression. A, the levels of p15INK4B and p16 mRNA were determined by qPCR in SPC-A1 and PC9 cells transfected with si-ANRIL and results 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 by qPCR and Western blot when PC9 cells were transfected with si-EZH2 or si-SUZ12. D, ANRIL expression levels in cell cytoplasm or 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.

Close modal

To further investigate whether ANRIL repress KLF2 and P21 expression through binding PRC2, we performed RIP analysis and the results showed that ANRIL could directly bind with EZH2 in PC9 cells (Fig. 6A). Furthermore, the results of ChIP assays showed that EZH2 could directly bind to KLF2 and P21 promoter region and mediate H3K27me3 modification (Fig. 6B). However, knockdown of ANRIL reduced EZH2 binding with KLF2 and P21 promoter (Fig. 6C). Finally, we detected the KLF2 expression in NSCLC tissues, and found that there is an inverse relationship between ANRIL and KLF2 expression (Fig. 6D). These data suggested that ANRIL promotes NSCLC PC9 cells proliferation is not dependent on regulation p15 expression, but also through silencing of KLF2 and P21 transcription.

Figure 6.

ANRIL could directly bind PRC2 and silence KLF2 and P21 transcription. A, RIP with rabbit monoclonal 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 IgG immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates were used as positive control. B and C, ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells, and IgG as a negative control; ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells treated with ANRIL siRNA (48 hours) or scrambled siRNA. D, analysis of the relationship between ANRIL expression and KLF2 mRNA level (ΔCt value) in 40 NSCLC tissues. The mean values and SDs were calculated from triplicates of a representative experiment.

Figure 6.

ANRIL could directly bind PRC2 and silence KLF2 and P21 transcription. A, RIP with rabbit monoclonal 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 IgG immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates were used as positive control. B and C, ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells, and IgG as a negative control; ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells treated with ANRIL siRNA (48 hours) or scrambled siRNA. D, analysis of the relationship between ANRIL expression and KLF2 mRNA level (ΔCt value) in 40 NSCLC tissues. The mean values and SDs were calculated from triplicates of a representative experiment.

Close modal

Silence of KLF2 is potentially involved in the oncogenic function of ANRIL

To investigate whether KLF2 is involved in the ANRIL-induced increase in NSCLC cell proliferation, we performed gain-of-function assays. The results of Western blot analysis showed that KLF2 expression was significantly upregulated in PC9 cells transfected with pCDNA–KLF2 compared with control cells (Fig. 7A). Meanwhile, MTT and colony formation assay results revealed that overexpression of KLF2 could impaired NSCLC cells proliferation (Fig. 7B). Moreover, flow-cytometry analysis indicated that increased KLF2 expression could induce NSCLC cells apoptosis (Fig. 7C). Furthermore, to determine whether ANRIL regulate NSCLC cell proliferation via repressing KLF2 expression, rescue assays were performed. PC9 cells were cotransfected with si-ANRIL and si-KLF2, and this was shown to rescue the decreased expression of ANRIL induced by knockdown of KLF2 (Fig. 7D). The results of MTT and colony formation assay results indicated that cotransfection could partially rescue si-ANRIL–impaired proliferation in PC9 cells (Fig. 7E). These data indicate that ANRIL promotes NSCLC cell proliferation through the downregulation of KLF2 expression.

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, the protein level of KLF2 in PC9 cells transfected with pCDNA–KLF2 was detected by Western blot analysis. B, MTT assays and colony-forming assays were used to determine the cell viability for pCDNA–KLF2-transfected PC9 cells. Values represent the mean ± SD from three independent experiments. C, apoptosis was 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 PC9 cells 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 cell viability 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.

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, the protein level of KLF2 in PC9 cells transfected with pCDNA–KLF2 was detected by Western blot analysis. B, MTT assays and colony-forming assays were used to determine the cell viability for pCDNA–KLF2-transfected PC9 cells. Values represent the mean ± SD from three independent experiments. C, apoptosis was 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 PC9 cells 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 cell viability 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.

Close modal

Recently, numerous pieces of evidence show that many lncRNAs are characterized and play important roles in cancer pathogenesis, suggesting that they could provide new insights into the biology of this disease. For example, increased lncRNA HOTTIP is associated with progression and predicts outcome in patients with hepatocellular carcinoma by regulating HOXA13 expression (22). However, the roles of lncRNAs in NSCLC are still not well documented, and one of these lncRNAs is metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which is a highly conserved nuclear lncRNA and a predictive marker for metastasis development in lung cancer (23). In our previous studies, we found that increased lncRNA HOTAIR–promoted NSCLC cells invasion and metastasis via regulating HOXA5 expression, and lncRNA BANCR overexpression could impaired NSCLC cells proliferation and metastasis by affecting epithelial–mesenchymal transition (24, 25).

In this study, we demonstrated that the expression of another lncRNA, ANRIL, is significantly upregulated in NSCLC tissues. Specifically, increased ANRIL expression appears to be a significant, independent predictive value for patients with NSCLC. Moreover, knockdown of ANRIL expression led to the significant inhibition of cell proliferation and the promotion of apoptosis both in vitro and in vivo. These findings suggest that ANRIL plays a direct role in the modulation of cell proliferation and NSCLC progression, and could be a useful novel prognostic or progression marker for NSCLC. As more and more lncRNAs are studied, many have been shown to function by binding with PRC2 and silencing downstream target genes that involved in multiple cancers, including NSCLC (26, 27). ANRIL has been reported to involve in cancer cells proliferation by silencing p15INK4 expression. In this study, we found that ANRIL is mostly located in cell nucleus and could directly bind with EZH2, a core subunit of PRC2, resulted in repressing KLF2 and P21 transcription. However, knockdown of ANRIL could not influence p15INK4 expression in NSCLC PC9 cells, which indicated that ANRIL contributed to NSCLC cell proliferation is not dependent on regulating p15INK4, but also could through silencing KLF2 and P21 transcription.

The Kruppel-like factor (KLF) family transcription factors, with Cys2/His2 zinc-finger domains, could function as suppressors or activators in a cell type and promoter-dependent manner and involve in cell differentiation and proliferation (28, 29). Some KLF members are emerging as tumor-suppressor genes due to their roles in the inhibition of proliferation, migration, and induction of apoptosis (30, 31). KLF2, as a member of KLF family, is diminished in many cancers and possesses tumor-suppressor features such as inhibition of cell proliferation mediated by KRAS (32–34). Moreover, there is evidence that showed that EZH2 could silence KLF2 expression and block the tumor-suppressor features of KLF2, which is partly mediated by p21 (21). Our results also showed that lncRNA ANRIL takes part in NSCLC cells' proliferation by epigenetic-silencing KLF2 and P21 transcription, and KLF2 inactivation further led to the decreased P21 expression. As more and more studies indicated that lncRNAs are often expressed in a spatial- or temporal-specific pattern, and more cell- and tissue-specific pattern. Our results also showed that even in NSCLC cells, lncRNA ANRIL could regulate different target genes in different cell lines, which suggested that lncRNA, especially ANRIL, can influence the same cell biologic function via regulating different target genes dependent on different cell lines.

To date, although only a small number of lncRNAs have been well characterized, they have been shown to regulate gene expression at various levels, including chromatin modification and posttranscriptional processing (35, 36). Here, the possible other targets and mechanisms that underlie such regulatory behaviors still remain to be fully understood despite our observation of ANRIL-induced NSCLC cell proliferation. In summary, the expression of ANRIL was significantly increased in NSCLC tissues, suggesting that its upregulation may be a negative prognostic factor for patients with NSCLC, indicative of poor survival rates, and a higher risk for cancer metastasis. We showed that ANRIL possibly regulates the proliferation ability of NSCLC cells, partially through its regulation of the KLF2 and P21, which indicated that lncRNAs contribute to different cancer cells biologic function maybe through regulating different target genes. Our findings further the understanding of NSCLC pathogenesis, and facilitate the development of lncRNA-directed diagnostics and therapeutics against cancers.

No potential conflicts of interest were disclosed.

Conception and design: F.-q. Nie, M. Sun, T.-p. Xu, K.-h. Lu, Y.-q. Shu

Acquisition 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, biostatistics, computational analysis): F.-q. Nie, J.-s. Yang, M. Xie, R. Xia, Y.-w. Liu, X.-h. Liu

Writing, review, and/or revision of the manuscript: F.-q. Nie, K.-h. Lu, Y.-q. Shu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.-b. Zhang

Study supervision: M. Sun, K.-h. Lu, Y.-q. Shu

The authors are very grateful to professor Xiongbin Lu for providing the ANRIL overexpression plasmid.

This work was supported by grants from the National Natural Scientific Foundation of China (81372397; to K.-h. Lu); (81301824; to X.-h. Liu); (81172140 and 81272532; to Y.-q. Shu). This work was also supported by Priority Academic Program Development of Jiangsu Higher Education Institutions (JX10231801). M. Sun was supported by a Jiangsu province ordinary university graduate student research innovation project for 2013 (CXZZ13_0562, JX22013265).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
R
,
Naishadham
D
,
Jemal
A
. 
Cancer statistics, 2013
.
CA Cancer J Clin
2013
;
63
:
11
30
.
2.
Spira
A
,
Ettinger
DS
. 
Multidisciplinary management of lung cancer
.
N Engl J Med
2004
;
350
:
379
92
.
3.
Wistuba
II
. 
Genetics of preneoplasia: lessons from lung cancer
.
Curr Mol Med
2007
;
7
:
3
14
.
4.
Verdecchia
A
,
Francisci
S
,
Brenner
H
,
Gatta
G
,
Micheli
A
,
Mangone
L
, et al
Recent cancer survival in Europe: a 2000–02 period analysis of EUROCARE-4 data
.
Lancet Oncol
2007
;
8
:
784
96
.
5.
International Human Genome Sequencing Consortium
. 
Finishing the euchromatic sequence of the human genome
.
Nature
2004
;
431
:
931
45
.
6.
Djebali
S
,
Davis
CA
,
Merkel
A
,
Dobin
A
,
Lassmann
T
,
Mortazavi
A
, et al
Landscape of transcription in human cells
.
Nature
2012
;
489
:
101
8
.
7.
Derrien
T
,
Johnson
R
,
Bussotti
G
,
Tanzer
A
,
Djebali
S
,
Tilgner
H
, et al
The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression
.
Genome Res
2012
;
22
:
1775
89
.
8.
Bartel
DP
. 
MicroRNAs: genomics, biogenesis, mechanism, and function
.
Cell
2004
;
116
:
281
97
.
9.
Fatica
A
,
Bozzoni
I
. 
Long non-coding RNAs: new players in cell differentiation and development
.
Nat Rev Genet
2014
;
15
:
7
21
.
10.
Geisler
S
,
Coller
J
. 
RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts
.
Nat Rev Mol Cell Biol
2013
;
14
:
699
712
.
11.
Loewer
S
,
Cabili
MN
,
Guttman
M
,
Loh
YH
,
Thomas
K
,
Park
IH
, et al
Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells
.
Nat Genet
2010
;
42
:
1113
7
.
12.
Mercer
TR
,
Dinger
ME
,
Mattick
JS
. 
Long non-coding RNAs: insights into functions
.
Nat Rev Genet
2009
;
10
:
155
9
.
13.
Wapinski
O
,
Chang
HY
. 
Long noncoding RNAs and human disease
.
Trends Cell Biol
2011
;
21
:
354
61
.
14.
Tsai
MC
,
Spitale
RC
,
Chang
HY
. 
Long intergenic noncoding RNAs: new links in cancer progression
.
Cancer Res
2011
;
71
:
3
7
.
15.
Spizzo
R
,
Almeida
MI
,
Colombatti
A
,
Calin
GA
. 
Long non-coding RNAs and cancer: a new frontier of translational research
?
Oncogene
2012
;
31
:
4577
87
.
16.
Zhang
EB
,
Kong
R
,
Yin
DD
,
You
LH
,
Sun
M
,
Han
L
, et al
Long noncoding RNA ANRIL indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of miR-99a/miR-449a
.
Oncotarget
2014
;
5
:
2276
92
.
17.
Kotake
Y
,
Nakagawa
T
,
Kitagawa
K
,
Suzuki
S
,
Liu
N
,
Kitagawa
M
, et al
Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene
.
Oncogene
2011
;
30
:
1956
62
.
18.
Chen
D
,
Zhang
Z
,
Mao
C
,
Zhou
Y
,
Yu
L
,
Yin
Y
, et al
ANRIL inhibits p15 through the TGFbeta1 signaling pathway in human esophageal squamous cell carcinoma
.
Cell Immunol
2014
;
289
:
91
6
.
19.
Yoon
JH
,
Abdelmohsen
K
,
Srikantan
S
,
Yang
X
,
Martindale
JL
,
De
S
, et al
LincRNA-p21 suppresses target mRNA translation
.
Mol Cell
2012
;
47
:
648
55
.
20.
Seward
S
,
Semaan
A
,
Qazi
AM
,
Gruzdyn
OV
,
Chamala
S
,
Bryant
CC
, et al
EZH2 blockade by RNA interference inhibits growth of ovarian cancer by facilitating re-expression of p21(waf1/cip1) and by inhibiting mutant p53
.
Cancer Lett
2013
;
336
:
53
60
.
21.
Taniguchi
H
,
Jacinto
FV
,
Villanueva
A
,
Fernandez
AF
,
Yamamoto
H
,
Carmona
FJ
, et al
Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer
.
Oncogene
2012
;
31
:
1988
94
.
22.
Quagliata
L
,
Matter
MS
,
Piscuoglio
S
,
Arabi
L
,
Ruiz
C
,
Procino
A
, et al
Long noncoding RNA HOTTIP/HOXA13 expression is associated with disease progression and predicts outcome in hepatocellular carcinoma patients
.
Hepatology
2014
;
59
:
911
23
.
23.
Gutschner
T
,
Hammerle
M
,
Eissmann
M
,
Hsu
J
,
Kim
Y
,
Hung
G
, et al
The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells
.
Cancer Res
2013
;
73
:
1180
9
.
24.
Liu
XH
,
Liu
ZL
,
Sun
M
,
Liu
J
,
Wang
ZX
,
De
W
. 
The long non-coding RNA HOTAIR indicates a poor prognosis and promotes metastasis in non–small cell lung cancer
.
BMC Cancer
2013
;
13
:
464
.
25.
Sun
M
,
Liu
XH
,
Wang
KM
,
Nie
FQ
,
Kong
R
,
Yang
JS
, et al
Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for non–small cell lung cancer and promotes metastasis by affecting epithelial–mesenchymal transition
.
Mol Cancer
2014
;
13
:
68
.
26.
Beckedorff
FC
,
Ayupe
AC
,
Crocci-Souza
R
,
Amaral
MS
,
Nakaya
HI
,
Soltys
DT
, et al
The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation
.
PLoS Genet
2013
;
9
:
e1003705
.
27.
Brockdorff
N
. 
Noncoding RNA and Polycomb recruitment
.
RNA
2013
;
19
:
429
42
.
28.
Black
AR
,
Black
JD
,
Azizkhan-Clifford
J
. 
Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer
.
J Cell Physiol
2001
;
188
:
143
60
.
29.
Kaczynski
J
,
Cook
T
,
Urrutia
R
. 
Sp1- and Kruppel-like transcription factors
.
Genome Biol
2003
;
4
:
206
.
30.
Shen
P
,
Sun
J
,
Xu
G
,
Zhang
L
,
Yang
Z
,
Xia
S
, et al
KLF9, a transcription factor induced in flutamide-caused cell apoptosis, inhibits AKT activation and suppresses tumor growth of prostate cancer cells
.
Prostate
2014
;
74
:
946
58
.
31.
Hsu
LS
,
Chan
CP
,
Chen
CJ
,
Lin
SH
,
Lai
MT
,
Hsu
JD
, et al
Decreased Kruppel-like factor 4 (KLF4) expression may correlate with poor survival in gastric adenocarcinoma
.
Med Oncol
2013
;
30
:
632
.
32.
Wu
J
,
Lingrel
JB
. 
KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1
.
Oncogene
2004
;
23
:
8088
96
.
33.
Wang
F
,
Zhu
Y
,
Huang
Y
,
McAvoy
S
,
Johnson
WB
,
Cheung
TH
, et al
Transcriptional repression of WEE1 by Kruppel-like factor 2 is involved in DNA damage-induced apoptosis
.
Oncogene
2005
;
24
:
3875
85
.
34.
Fernandez-Zapico
ME
,
Lomberk
GA
,
Tsuji
S
,
DeMars
CJ
,
Bardsley
MR
,
Lin
YH
, et al
A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth
.
Biochem J
2011
;
435
:
529
37
.
35.
Ponting
CP
,
Oliver
PL
,
Reik
W
. 
Evolution and functions of long noncoding RNAs
.
Cell
2009
;
136
:
629
41
.
36.
Nagano
T
,
Fraser
P
. 
No-nonsense functions for long noncoding RNAs
.
Cell
2011
;
145
:
178
81
.