MOLECULAR CARCINOGENESIS

A Critical Role for the Long Non-Coding RNA GAS5 in Proliferation and Apoptosis in Non-Small-Cell Lung Cancer Xuefei Shi,1 Ming Sun,2 Hongbing Liu,1 Yanwen Yao,1 Rong Kong,2 Fangfang Chen,1 and Yong Song1* 1 2

Department of Respiratory Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, China

In more recent years, long non-coding RNAs (lncRNAs) have been investigated as a new class of regulators of cellular processes, such as cell growth, apoptosis, and carcinogenesis. Although lncRNAs are dysregulated in numerous cancer types, limited data are available on the expression profile and functional role of lncRNAs in non-small cell lung cancer (NSCLC). In the present study, we determined the expression pattern of the growth arrest-specific transcript 5 (GAS5) in 72 NSCLC specimens by qRT-PCR and assess its biological functions in the development and progression of NSCLC. The results revealed that GAS5 expression was down-regulated in cancerous tissues compared to adjacent noncancerous tissues (P < 0.05) and was highly related to tumor size and TNM stage (P < 0.05). This correlation between GAS5 and clinicopathological parameters indicates that GAS5 might function as a tumor suppressor. Furthermore, GAS5 overexpression increased tumor cell growth arrest and induced apoptosis in vitro and in vivo. Meanwhile, siRNA-mediated knockdown of GAS5 promoted tumor cell growth. Importantly, through western blot analysis, we found that ectopic expression of GAS5 significantly up-regulated p53 expression and down-regulated transcription factor E2F1 expression. Taken together, these findings suggest that GAS5 is a tumor suppressor in NSCLC, and the action of GAS5 is mediated by p53-dependent and p53-independent pathways. GAS5 could serve as a potential diagnostic marker for NSCLC and may be a novel therapeutic target in patients with NSCLC. © 2013 Wiley Periodicals, Inc. Key words: long non-coding RNAs; GAS5; NSCLC; proliferation; apoptosis

INTRODUCTION Lung cancer remains the leading cause of cancerrelated death worldwide and is expected to account for 28% of all male cancer deaths and 26% of all female cancer deaths in 2013 [1,2]. There are two main types of lung cancer: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), which accounts for 85% of lung cancer cases [3]. The crucial reason for high mortality is sustained proliferation and the tumor’s metastatic potential [4]. Therefore, a better understanding of the molecular mechanisms underlying tumor cell proliferation and apoptosis is needed for more efficient NSCLC management [5,6]. In the past few decades, numerous molecular epidemiological studies have indicated that the mutations in certain protein-coding genes (TP53, EGFR, KRAS) play an important role in the pathogenesis of NSCLC [7,8]. However, recent advances have revealed that another important type of genes, non-coding genes, may also be emerging as new players in the cancer paradigm [9]. Long non-coding RNA is an RNA molecule that is longer than 200 nucleotides and is not translated into a protein [10]. Although these long non-coding transcripts were once considered to be simply transcriptional “noise” or cloning artifacts [11], increasing evidence has suggested that lncRNAs participate in a ß 2013 WILEY PERIODICALS, INC.

Abbreviations: 5-aza-CdR, 5-aza-20 -deoxycytidine; AF-1, activation function 1; cIAP2, the cellular inhibitor of apoptosis 2; DMEM, Dulbecco's modified Eagle's medium; E2F1, Transcription factor E2F1; EGFR, epidermal growth factor receptor protein; ER, estrogen receptor; FBS, fetal bovine serum; GAS5, the growth arrest-specific transcript 5; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HBE, human bronchial epithelial cell; KRAS,V-Ki-ras2, Kirsten rat sarcoma viral oncogene homolog; lncRNAs, long noncoding RNAs; MALAT 1, metastasis associated lung adenocarcinoma transcript 1; MEG3, maternally expressed 3; NF-YA, Nuclear transcription factor Y subunit alpha; NRs, nuclear receptors; NSCLC, non-small-cell lung cancer; PANDA, p21 associated ncRNA DNA damage activated; PR, progestrone receptor; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; SCLC, small-cell lung cancer; SD, standard deviation; SDS–PAGE, SDS-polyacrylamide gel; siRNAs, small interfering RNAs; SRA, steroid receptor RNA activator; TP53, tumor protein p53; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; SGK1, the serum/glucocorticoid-regulated kinase 1; wt, wildtype. The authors have no conflict of interest to declare. Grant sponsor: National Natural Science Foundation of China; Grant number: 81170064; Grant sponsor: Natural Science Foundation of China; Grant number: 81302032 *Correspondence to: Department of Respiratory Medicine, Jinling Hospital, Medical School of Nanjing University, 305 East Zhongshan Road, Nanjing, Jiangsu Province 210002, China. Received 28 August 2013; Revised 22 November 2013; Accepted 26 November 2013 DOI 10.1002/mc.22120 Published online in Wiley Online Library (wileyonlinelibrary.com).

2

SHI ET AL.

spectrum of biological function, including cell differentiation, cell fate determination, proliferation, and migration [12,13]. In this regard, highlighting the potentially widespread functional roles of lncRNAs in human cancer is important. SRA (steroid receptor RNA activator), a coactivator for nuclear receptors (NRs), fulfills its activation function through the AF-1 domain of the nuclear receptor [13]. Increased SRA expression may alter ER/PR activity, leading to cell proliferation and breast tumorigenesis [14]. In addition to SRA, another example of a lncRNA affecting cell growth control is PANDA (p21 associated ncRNA DNA damage activated). Further experiments have showed that PANDA can interact with the transcription factor NF-YA, resulting in the down-regulation of pro-apoptotic genes. Accordingly, PANDA depletion has been shown to markedly sensitize human fibroblasts to doxorubicin-induced apoptosis [15]. Unfortunately, the emerging functional role of lncRNAs in NSCLC remains largely unknown. The growth arrest-specific transcript 5 (GAS5) was originally isolated from a subtraction cDNA library identifying potential tumor suppressor genes enriched during growth arrest by Schneider et al. [16]. GAS5 is encoded at 1q25 and is approximately 630 nt in length. The human GAS5 encoding gene (gas5), one of the 50 -terminal oligopyrimidine (50 TOP) class genes, comprises 12 exons, which have little proteincoding potential and encode 10 box C/D snoRNAs within its introns [17–19]. Hence, it had previously been assumed that any important biological function of gas5 must be mediated through introns [19]. However, Kino et al. [20] found that mature GAS5 lncRNA (exon 12-derived sequence) is up-regulated during growth arrest induced by serum starvation or the lack of growth factors. They found that GAS5 was able to compete with DNA GREs for binding to the DNA-binding domain of the glucocorticoid receptor (GR), leading to the suppression of the glucocorticoidmediated induction of several response genes. Thus, lncRNA GAS5, which does not contain introns, may also be involved in many novel, unexpected cellular functions. Furthermore, earlier experiments have demonstrated that GAS5 overexpression leads to a slower cell cycle and an increase in apoptosis [21]. Together, these findings are consistent with GAS5 playing an essential role in normal growth arrest and apoptosis. Yet, the expression profile and biological role of GAS5 and its mechanism in NSCLC remains largely unknown. In the present study, we not only found that GAS5 expression was significantly down-regulated in NSCLC tissue compared to adjacent normal lung tissue but also showed that the overexpression of GAS5 transcript, which comprises 12 exons, induced growth arrest and apoptosis in human lung cancer cell lines. Our results suggest that decreased GAS5 expression may be important in NSCLC carcinogenesis. Molecular Carcinogenesis

MATERIALS AND METHODS Patients and Tissue Samples Paired NSCLC and adjacent normal lung tissue were obtained from 72 patients who underwent primary surgical resection of a NSCLC in the Department of Thoracic Surgery, Jinling Hospital, Nanjing University School of Medicine, China, between November 2011 and September 2012. No patient had received preoperative adjuvant therapy, and the clinicopathologic characteristics of the patients with NSCLC are summarized in Table 1. Following surgical removal, the tissue samples were immediately frozen in liquid nitrogen and stored at 808C until RNA extraction. The study protocol was approved by the Institutional Review Board of Nanjing University, and all of the participants signed an informed consent form. Cell Culture Seven NSCLC cell lines (A549, H1650, H1299, H1975, SK-MES, SPC-A1, and HBE) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). A549, H1650, H1299, H1975, SK-MES, and HBE cells were maintained in RPMI Medium 1640 basic medium (GIBCO-BRL; Invitrogen, Carlsbad, CA), and SPCA1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL; Invitrogen), supplemented with heat-inactivated 10% fetal bovine serum (FBS) and antibiotics at 378C in a humidified incubator with 5% CO2. RNA Isolation and Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) Analyses Total RNA was extracted from frozen tissue or cultured cells using TRIZOL reagent (Invitrogen), according to the manufacturer’s instructions. For GAS5 detection, the isolated RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara, Dalian, China). GAS5 lncRNA was quantified by qRT-PCR using SYBR Premix Ex Taq II (Perfect Real Time; TaKaRa) according to the manufacturer’s instructions. The gene-specific primers were as follows: GAPDH sense 5’-GTCAACGGATTTGGTCTGTATT-30 , reverse 50 -AGTCTTCTGGGTGGCAGTGAT30 ; GAS5 sense, 50 -CTTGCCTGGACCAGCTTAAT-30 , reverse 50 -CAAGCCGACTCTCCATACCT-30 ; SGK1 sense 50 -CTATGCTGCTGAAATAGC-30 , reverse 50 GTCCGAAGTCAGTAAGG-30 ; cIAP2 sense 50 TCTAGTGTTCTAGTTAATCC-30 , reverse 50 -ACCACTTGGCATGTTGAACC-30 . GAPDH was used for normalization. The PCR reaction was conducted at 958C for 30 s followed by 40 cycles of 958C for 5 s and 608C for 34 s in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA). All of the qRT-PCRs were performed in duplicate. The relative quantification of GAS5 expression was calculated using the 2-DDCT method relative to GAPDH.

3

GAS5 REGULATES PROLIFERATION AND APOPTOSIS OF NSCLC

Table 1. Correlation Between GAS5 Expression and Clinicopathological Parameters of NSCLC. Relative GAS5 expression Clinicopathological parameters Age (years) 65 >65 Gender Male Female Differentiation Well, moderate Poor Tumor size (maximum diameter cm) 5 cm >5 cm Primary location Left lung Right lung Histology type Adenocarcinoma Squamous carcinoma Lymph node metastasis Positive Negative TMN stage I II/III/IV a

No. of cases

Low

High

50 22

31 15

19 7

55 17

36 10

19 7

48 24

30 16

18 8

36 36

18 28

18 8

36 36

22 24

14 12

38 34

22 24

16 10

33 39

22 24

11 15

23 49

10 36

13 13

P-valuea 0.615 0.619 0.729 0.014b 0.624 0.263 0.652 0.013b

Chi-squared test. P < 0.05.

b

Treatment of H1650 and A549 Cells With 5-aza-2-deoxycytidine (5-aza-CdR) H1650 and A549 cells (2.5  105) were cultured on six-well plates on day 0 and exposed to 0, 5 or 10 mM 5-aza-CdR (Sigma–Aldrich, St. Louis, MO) from day 1 to 3. The cells treated with 5-aza-CdR were harvested on day 3 and used to detect GAS5 expression by qRT-PCR. Plasmid DNA Transfection The GAS5 sequence was synthesized according to the full-length GAS5 sequence lacking a poly A tail (based on the GAS5 sequence, NR_002578, in NCBI) and then subcloned into a pCDNA3.1 vector (Invitrogen, Shanghai, China). The pCDNA-GAS5 or empty vector was transfected into H1650 and A549 cells cultured on six-well plates using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions. The empty pcDNA3.1 vector was used as the control. Cells were harvested 48 h after transfection, and the total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. QRT-PCR analysis was used to assess for GAS5 overexpression. RNA Interference by siRNA The sequences of the siRNAs (small interfering RNAs) were the following: control siRNA sense 50 Molecular Carcinogenesis

GUACCUGACUAGUCGCAGAAG-30 , antisense 50 GUACCUGACUAGUCGCAGAAG-30 ; GAS5 siRNA sense 50 -CUUGCCUGGACCAGCUUAAUU-30 , antisense 50 -UUAAGCUGGUCCAGGCAAGUU-30 ; SUZ12 siRNA sense 50 -GCCGCAAACUUUAUAGUUUACUCAA-30 , antisense 50 -UUGAGUAAACUAUAAAGUUUGCGGC-30 ; EZH2 siRNA sense 50 -GAGGUUCAGACGAGCUGAU-30 , antisense 50 -AUCAGCUCGUCUGAACCUC-30 . The GAS5 and control siRNAs were transfected into SPC-A1 cells, as well as the SUZ12 and EZH2 siRNAs were transfected into A549 cells cultured on six-well plate using Lipofectamine2000 (Invitrogen, Shanghai, China), according to the manufacturer’s instructions. The cells were harvested 24 h after transfection, and the total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The specific silencing of GAS5 expression was assessed using qRT-PCR. Determination of Cell Viability and Colony Formation Assay Cell proliferation was monitored using the Cell Proliferation Reagent Kit I (MTT; Roche Applied Science). The pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells (3,000/well) and control siRNA- or GAS5 siRNA-transfected SPC-A1 cells (3,000/ well) were allowed to grow in 96-well plates. Cell proliferation was documented every 24 h following

4

SHI ET AL.

the manufacturer’s protocol. All of the experiments were performed in quadruplicate. For the cells to form colonies, a total of 700 pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells and control siRNA- or GAS5 siRNA-transfected SPC-A1 cells were placed onto a fresh six-well plate and maintained in media containing 10% FBS, replacing the medium every 4 d. After 2 wk, the colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma, St. Louis, MO). The visible colonies were manually counted. Triplicate wells were assessed for each treatment group. Flow-Cytometric Analysis of Apoptosis and Cell Cycle The pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells were cultured in six-well plates for 48 h. The cells were harvested by trypsinization. Following double staining with FITC-annexin V and propidium iodide (PI), the cells were analyzed using flow cytometry (FACScan; BD Biosciences, San Jose, CA) and equipped with a CellQuest software (BD Biosciences). The cells were categorized into early apoptotic cells, late apoptotic cells, dead cells, and viable cells. The percentage of apoptotic cells were compared to the control group from each experiment. All of the samples assayed were from triplicate experiments. For the flow-cytometric analysis of the cell cycle, the cells were stained with PI using the CycleTESTTM PLUS DNA reagent kit (BD Biosciences) following the protocol and analyzed by FACScan. The percentage of cells in the S, G0/G1, and G2/M phases were counted and compared. Transwell Assay Transwell assays were performed using polycarbonate transwell filters (Corning Costar Corp, Cambridge, MA) placed over the bottom chambers, which were filled with culture medium containing 10% FBS. A sample of A549 cells (5  104) that were treated for 48 h with pCDNA-GAS5 or empty vector was suspended in RPMI1640 medium and seeded into the upper chamber. After 24 h of culture at 378C, we removed the upper layer of cells before visualization, and the cells on the lower surface were fixed in paraformaldehyde and stained with 0.1% crystal violet (Sigma). The cells on the underside of the filter were counted in five random fields and photographed. For each experiment, three independent filters were analyzed. Terminal Deoxynucleotidyl Transferase dUTP Nick end Labeling (TUNEL) The pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells were cultured in six-well plates for 48 h. They were then fixed, and the apoptotic cells were labeled using the in situ cell death detection kit, Fluorescein (Roche Applied Science), according to the manufacturer’s instructions. The apoptotic cells were detected using an optical microscope and counted in three different experiments. Molecular Carcinogenesis

Western Blotting The pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells were lysed using a lysis buffer that contained the mammalian protein extraction reagent RIPA (Beyotime China), a protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF (Roche). The protein concentration was determined using a Bio-Rad protein assay kit. The samples that contained 50-mL protein from two different cell lines were electrophoresed on a 10% SDS–polyacrylamide gel (SDS–PAGE) and transferred onto 0.22-mm nitrocellulose membranes (Sigma–Aldrich) and incubated with specific antibodies. Specific bands were detected using an ECL chromogenic substrate. Protein expression was analyzed using densitometry (Quantity One Software; Bio-Rad, Hercules, California). GAPDH was used as a control. Additionally, anti-p53 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-p21 and anti-E2F1 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-GAPDH antibody was purchased from Sigma-Aldrich (USA). Tumor Formation Assay in a Nude Mouse Model Age (4 wk)- and sex (male)-matched nude mice were used for the tumor formation assay. All of the mice were BALB/c background. The animal care and experimental procedures were approved by the Model Animal Research Center of Jingling Hospital and conducted according to Institutional Animal Care and User guidelines. A549 cells were transfected with pCDNA-GAS5 or empty vector were cultured in sixwell plates for 48 h. Then, the cells were washed with PBS and resuspended at a concentration of 2  107 cells/mL. Each mouse was injected on the right side of the posterior flank with 2  106 suspended cells. Tumor growth was measured by calipers every 3 d. The tumors were removed from all of the animals after 15 d, and the subcutaneous growth of each tumor was examined. The tumor volumes were calculated using the equation V ¼ 0.5  D  d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). All of the surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Statistical Analysis Statistical analysis was performed using SPSS version 18 software (Chicago, IL). Two-tailed Student’s t-test and one-way ANOVA were performed to analyze the in vitro and in vivo data. The results are expressed as the mean  SD. A P-value less than 0.05 was considered to be statistically significant. RESULTS Down-Regulation of GAS5 in NSCLC Tissues To ascertain whether lncRNA GAS5 was differentially expressed in the NSCLC tissues, a total of 72

GAS5 REGULATES PROLIFERATION AND APOPTOSIS OF NSCLC

paired clinical NSCLC tissues and adjacent normal tissues were analyzed for GAS5 expression using qRT-PCR. GAS5 expression was significantly downregulated (1.73-fold) in clinical NSCLC specimens (T) compared to adjacent normal lung tissues (N) (P < 0.05; Figure 1A). Furthermore, we evaluated the correlation of GAS5 expression with clinicopathological parameters (i.e., stage, maximum diameter) to assess its clinical significance. As presented in Figure 1B and C and Table 1, larger tumors, which represent a higher tumor burden, or more advanced tumors had lower GAS5 expression. Nonetheless, there was no significant relationship between GAS5 expression and other clinical characteristics, such as lymph node metastasis (Figure 1D), differentiation, histology type or gender (P > 0.05; Table 1). These analyses demonstrate that GAS5 may be a good diagnostic biomarker for early-stage NSCLC. GAS5 Expression in NSCLC Cells To understand the role of GAS5 in the development of NSCLC, we used qRT-PCR analysis to assess GAS5 expression in NSCLC cell lines. We found that GAS5 expression was at a comparatively low level in 5 lung

5

cancer cell lines, including A549, H1650, H1299, H1975, and SK-MES, compared to human bronchial epithelial cells, whereas GAS5 expression in SPC-A1 was slightly higher than in HBEs (Figure 2A). Methylation in the promoter region of lncRNAs has been reported to affect the transcriptional activation of many lncRNAs [22]. Additionally, bioinformatics analysis has shown that there are CpG islands in the GAS5 promoter regions. Thus, to investigate the potential mechanism leading to decreased GAS5 expression, we focused on the methylation of CpG islands in the GAS5 promoter regions. We exposed H1650 and A549 cells to 5-aza-2-deoxy-cytidine (5aza-CdR) at different concentrations and analyzed the GAS5 expression in these cells using qRT-PCR. GAS5 expression was significantly increased after treatment (Figure 2B and C). Additionally, Sun and colleagues demonstrate that the histone modification in lncRNAs promoter region could also affect the expression of lncRNAs [23]. Hence, we chose the polycomb repressive complexes 2 (PRC2) which has intrinsic histone methyltransferase (HMT) activities to detect the role of histone methylation in impacting the expression of GAS5. However, siRNA-mediated knockdown of SUZ12 or EZH2, two main components of PRC2, indicated that there was no statistical correlation between histone methylation and GAS5 expression (Figure 2D). Therefore, DNA methylation may partially contribute to the down-regulation GAS5 expression in NSCLC tissues and cell lines. With the purpose of manipulating the GAS5 level in NSCLC cells, the pCDNA-GAS5 vector was transfected into H1650 and A549 cells. After 48 h of transfection, GAS5 expression was substantially up-regulated, and the efficiency of transfection for H1650 and A549 cells was 71- and 56-fold, respectively, compared to the respective control cells (Figure 2E). Meanwhile, we used small interfering RNAs (siRNAs) to down-regulate endogenous GAS5 expression in the SPC-A1 cells, and 80% down-regulation for the siRNAs targeting GAS5 was confirmed by qRT-PCR (Figure 2F). The Effect of GAS5 on Cell Proliferation in the NSCLC Cell Lines

Figure 1. Relative GAS5 expression in NSCLC tissues and its clinical significance. (A) qRT–PCR analysis of the relative GAS5 expression in NSCLC tissues (n ¼ 72) and in paired adjacent normal tissues (n ¼ 72). GAS5 expression was normalized to GAPDH expression. The data are presented as a fold-change in the tumor tissue relative to the normal tissue. (B) GAS5 expression was significantly lower in larger tumors. (C) GAS5 expression was significantly lower in patients with an advanced clinical stage than those with an early clinical stage. (D) There was no significant relationship between the GAS5 expression profile and the presence of lymph node metastasis.  P < 0.05,  P < 0.01.

Molecular Carcinogenesis

Because human lncRNAs participate in a spectrum of biological processes, we examined the impact of GAS5 overexpression in the NSCLC cell lines. Compared to the empty vector-transfected cells, transfection with pCDNA-GAS5 resulted in a significant decrease in H1650 and A549 cell viability as monitored by an MTT (Figure 3A and B). In addition, the long-term survival assay showed that GAS5 overexpression also greatly attenuated the colony-forming ability of the population (Figure 3C and D). Additionally, the growth inhibition was accompanied by a corresponding decrease in the proportion of cells in the S phase and a clear increase in the proportion of cells in G1 (Figure 3E and F).

6

SHI ET AL.

Figure 2. GAS5 expression in NSCLC cells. (A) qRT-PCR results demonstrating GAS5 expression in NSCLC cell lines (A549, H1650, H1299, H1975, SK-MES, SPC-A1) compared to human bronchial epithelial cells (HBEs). (B and C) qRT-PCR measurement of GAS5 expression following the treatment of H1650 and A549 cells with 5-aza-CdR. (D) qRT-PCR analysis of GAS5 expression following the treatment of A549 cells with si-EZH2 and si-SUZ12. (E) GAS5 expression, measured by qRT-PCR, following the treatment of H1650 and A549 cells with pCDNA-GAS5 or empty vector. (F) Using qRT-PCR, GAS5 expression was measured following the treatment of SPC-A1 cells with si-RNA GAS5 by qRT-PCR analyses. Error bars represent SD.  P < 0.05,  P < 0.01.

GAS5 Overexpression Induced Apoptosis in the H1650 and A549 Cells Apoptosis is major mechanism leading to controlled cell death, and defects in apoptosis can cause tumorigenesis [13,24]. To gain insight into the important role of GAS5 in regulating apoptosis in the NSCLC cell lines, H1650 and A549 cells were transfected with pCDNA-GAS5 and empty vector. Cells were harvested 48 h following transfection and analyzed using flow cytometry. As shown in Figure 4A and B, GAS5 overexpression significantly induced apoptosis, especially early apoptosis. Consistently, microscopic analysis of TUNEL staining of H1650 and A549 cells also showed that the number of apoptotic cells was greater in the GAS5 overexpression cells than the controls (Figure 4C and D, respectively). Both flow cytometric analysis and TUNEL staining suggested that GAS5 has a critical effect on lung cancer cell apoptosis. Taken together, GAS5 expression is both important and necessary for normal growth arrest and apoptosis in the NSCLC cell lines. Increased GAS5 Expression Had No Effect on Migration and Invasion GAS5 is ubiquitously expressed during growth arrest and was originally identified in growth-arrested mouse NIH 3T3 fibroblasts [16]. Previous studies have emphasized the correlation between GAS5 expression and growth arrest and apoptosis. In sharp contrast, there are few studies demonstrating the influence of GAS5 on cellular migration and invasion in NSCLC Molecular Carcinogenesis

cell lines. Thus, we selected A549 cells as the experimental model and used in vitro Boyden chamber assays with or not with matrigel model. After 24 h, the results were obtained. GAS5 overexpression had no effect on migration and invasion (Figure 5A–C), compared to the control cells. This finding supported the clinicopathological parameter analysis. GAS5 Knockdown Mildly Promoted Proliferation in SPC-A1 Cells GAS5 expression has been widely acknowledged to be lower in tumor tissues and proliferative cells. Importantly, our clinical data indicated that GAS5 expression was inversely related to NSCLC progression. Thus, we used siRNAs to down-regulate endogenous GAS5 expression in the SPC-A1 cell line. An MTT assay as described previously was performed to monitor the effect of GAS5 siRNA on cell growth and proliferation at different times, and a colonyforming assay was also performed. The cell proliferation rate in the cultures treated with the GAS5 siRNA was barely distinguishable from the control cells at 96 h (Figure 5D). Additionally, the GAS5 siRNA also mildly protected the colony-forming ability compared to negative control (Figure 5E and F). The observations made with the siRNA indicated that lower GAS5 expression may contribute to NSCLC progression. GAS5 Overexpression Suppressed NSCLC Tumor Growth In Vivo Although GAS5 functioning as tumor suppressor gene has been demonstrated in various cell lines,

GAS5 REGULATES PROLIFERATION AND APOPTOSIS OF NSCLC

7

Figure 3. The effect of GAS5 on NSCLC cell proliferation and cell cycle in vitro. H1650 and A549 cells were transfected with pCDNA-GAS5 or empty vector, respectively. (A and B) An MTT assay was performed to determine the proliferation of pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells. (C and D) A colony-forming growth assay was performed to determine the proliferation of pCDNA-GAS5- or empty vector-transfected H1650 and A549 cells. The colonies were counted and captured. (E and F) Cell-cycle analysis was performed 48 h following the treatment H1650 and A549 cells with pCDNA-GAS5 or empty vector. The DNA content was quantified by flowcytometric analysis. The data represent the mean  SD from three independent experiments.  P < 0.05,  P < 0.01.

there was little direct evidence of the effect of GAS5 in vivo. To further provide in vivo evidence for the antioncogenic role of GAS5 in NSCLC, we used a xenograft mouse model. A total of 12 BALB/c mice were subcutaneously injected with A549 cells transfected with pCDNA-GAS5 (6 mice) or empty vector (6 mice) randomly. Three days after injection, all of them developed detectable tumors. Compared to the control treatment, GAS5 overexpression treatment dramatically decreased tumor growth, which was demonstrated by significantly reduced tumor size and weight (Figure 6A–C). The up-regulation of GAS5 in tumors after pCDNA-GAS5 transfection was confirmed by qRT-PCR analysis (Figure 6D). Thus, GAS5 Molecular Carcinogenesis

overexpression reduces the growth of established NSCLC xenografts. In addition, the HE staining showed the typical characteristics of tumor cells, and the proliferation index Ki67 determined by immunohistochemical staining significantly decreased in the GAS5-transfected tumors (Figure 6E). Taken together, these results clearly showed that GAS5 inhibited NSCLC development through diverse cellular processes. p53 and E2F1 Are Key Downstream Mediators of GAS5 Because an earlier study reported the involvement of GAS5 in suppressing the glucocorticoid-mediated induction of several responsive genes by acting as a

8

SHI ET AL.

Figure 4. The effect of GAS5 on NSCLC cell apoptosis in vitro. H1650 and A549 cells were transfected with pCDNA-GAS5 or empty vector, respectively. (A and B) The percentage of apoptotic cells was determined by flow-cytometric analysis. The data represent the mean  SD from three independent experiments. (C and D) TUNEL demonstrated apoptosis in H1650 and A549 cells transfected with pCDNA-GAS5 or empty vector.  P < 0.05 and  P < 0.01.

decoy “glucocorticoid response element (GRE)” in HeLa cells [2]. We examined the expression of the cellular inhibitor of apoptosis 2 (cIAP2) and the serum/glucocorticoid-regulated kinase 1 (SGK1), Molecular Carcinogenesis

which were mentioned in the earlier study. Unexpectedly, the qRT-PCR results revealed that GAS5 overexpression or GAS5 knockdown did not statistically affect the expression of these anti-apoptotic

GAS5 REGULATES PROLIFERATION AND APOPTOSIS OF NSCLC

9

Figure 5. The effect of GAS5 on NSCLC cells migration and invasion and GAS5 suppression promote NSCLC cell proliferation. (A–C) Representative images are shown of a Boyden chamber assay for the effect of GAS5 on A549 cell migration and invasion. (D) The proliferation of si-GAS5-transfected SPC-A1 cells was determined by an MTT assay. (E and F) A colony-forming growth assay was performed to determine the proliferation of si-GAS5 transfected SPC-A1 cells. The data represent the mean  SD from three independent experiments.  P < 0.05,  P < 0.01.

genes (Figure 7A and B), possibly because the behavior of the NSCLC cell lines differ from the HeLa cells. To further investigate the underlying molecular mechanisms responsible for the growth arrest and

apoptosis induced by GAS5 in NSCLC, we focused on associated signaling pathways. Notably, p53 has been well established as a key factor for controlling cellular senescence, cell cycle arrest and apoptosis [3]. Hence,

Figure 6. GAS5 inhibits tumor growth in a xenograft mouse model. (A) Tumor growth curve. A549 cells were transfected with pCDNA-GAS5 or empty vector and then injected into nude mice as described in the Materials and Methods Section. Tumor growth was measured from day 3 after injecting tumor cells. The error bars represent the standard deviation (SD). (B) Total number of tumors after removal from the mice. (C) Tumor weight when the tumors were harvested. The data represent the mean  SD.  P < 0.05 and  P < 0.01. (D) qRT-PCR analyses indicated that GAS5 expression is significantly increased in vivo. The data represent the mean  SD  P < 0.05 and  P < 0.01. (E) Representative images (200) of HE staining and immunohistochemistry of the tumor. The IHC showed an upregulation of GAS5 decreased the proliferation index Ki67.

Molecular Carcinogenesis

10

SHI ET AL.

Figure 7. p53 and E2F1 are key downstream mediators of GAS5. (A and B) The expression of cellular inhibitor of apoptosis 2 (cIAP2) and the serum/glucocorticoid-regulated kinase 1 (SGK1) was measured using qRT-PCR. (C–F) Expressions of p53, p21, E2F1, and Cyclin D1 were presented and evaluated in C and F, D and G, E and F, respectively. The results are from three independent experiments. GAPDH protein expression was used as an internal control.  P < 0.05;   P < 0.01.

we hypothesized that the GAS5 functions were mediated by the p53-dependent pathway. To confirm this possibility, we transfected the expression constructs for GAS5 into A549 cells that contained wildtype (wt) p53. As expected, transfection of pCDNAGAS5 led to a significant increase in p53 protein expression (Figure 7C and F). Meanwhile, we also assayed for changes in the protein expression of p21, an important p53 target gene. We found that the transfection of pCNDA-GAS5 into A549 elevates p21 protein expression compared to the control cells (Figure 7C and F). However, p53 and p21 mRNA levels remained unaltered in the GAS5 overexpression cells as confirmed by qRT-PCR (data not shown). These data suggest that GAS5 up-regulated p53 expression through post-transcriptional regulation and then stimulated expression of the endogenous target gene p21. Because the pathway mentioned above requires the presence of wide-type p53, the action of GAS5 as a tumor suppressor may be mediated by p53-independent pathways in H1650 cells without p53. This conclusion prompted us to examine other important Molecular Carcinogenesis

downstream mediators of GAS5. Indeed, we found that the expression of the transcription factor E2F1 was down-regulated in the pCDNA-GAS5-transfected H1650 cells (Figure 7D and G). A previous study showed that GAS5 was able to increase the proportion of cells in the G1 phase and was accompanied by a corresponding decrease in the proportion of cells in the S phase. Consistently, cyclinD1 protein expression was also disrupted in the GAS5-transfected NSCLC cells compared to the control cells (Figure 7E and H), whereas, the mRNA expression of E2F1 or cyclinD1 was not changed in the GAS5overexpressed A549 cells which was analysed by qRTPCR (data not shown). Taken together, both the p53 pathway and E2F1-mediated transcriptional activity play important roles in the cellular responses induced by the lncRNA GAS5. DISCUSSION In 2012, the GENCODE lncRNA catalog consists of 14,880 transcripts grouped into 9,277 gene loci in the human genome [25]. They are dynamically expressed

GAS5 REGULATES PROLIFERATION AND APOPTOSIS OF NSCLC

in differentiation-, tissue- and cell type-specific patterns [25,26]. Recent studies have identified a large number of lncRNAs involved in the development of human diseases, especially tumors [10,27,28]; however, the biological function of the vast majority remains unknown. In the present study, our attention focused on the lncRNA GAS5. We found that GAS5 was down-regulated in non-small cell lung cancer compared to the adjacent normal lung tissues. More importantly, GAS5 expression correlated with tumor size and clinical stage of the NSCLC. This report is the first direct investigation of the relationship between GAS5 expression and NSCLC. Consistently, earlier studies showed that GAS5 and/or its snoRNAs are down-regulated in many cancers, such as breast cancer, head and renal cell carcinoma, prostate cancer, and glioblastoma multiforme [29–31]. Taken together, the observations from this study indicate that GAS5 may function as a tumor suppressor in human tumor progression. Additionally, the result of overexpression and lossof-function analyses showed that GAS5 expression is both necessary and sufficient for growth arrest and apoptosis in the NSCLC cell lines. Similarly, our in vivo data also showed that the average tumor weight or volume was significantly lower for the mice injected with GAS5-transfected A549 cells compared to the control. This observation is of particular interest because there are few studies which have examined the biological function of GAS5 in vivo. GAS5 overexpression can effectively suppress the progression of NSCLC xenografts. These results have identified an important role for GAS5 in NSCLC and clarified the potential application of GAS5 in NSCLC. Although GAS5 has been shown to have crucial biological roles and is deregulated in various human cancers, the precise regulatory mechanism for GAS5 expression remains largely unknown. An earlier study demonstrated that the nonsense-mediated RNA decay (NMD) pathway can regulate the transcript levels of GAS5 [32]. More recently, Zhang and colleague found that miR-21 is capable of suppressing the lncRNA GAS5. Furthermore, GAS5 can also repress miR-21 expression [33]. However, in this study, via bioinformatics analysis, we found that there were CpG islands in the GAS5 promoter regions. Additionally, the upregulation of GAS5 expression after treatment with 5aza-2-deoxy-cytidine indicated that epigenetic regulation may also contribute to abnormal GAS5 expression. Moreover, as to the exact downstream factors for the tumor suppressor, p53 plays a central role in modulating many tumor suppressors [34–38]. Its activation leads to replication senescence, cell cycle arrest, or apoptosis by regulating the expression of its important target genes [39–41]. Hence, genetic inactivation of p53 has been correlated with various human cancers [42]. Moreover, an increasing number of studies have demonstrated the involvement of lncRNAs in the p53 transcriptional network [43]. For Molecular Carcinogenesis

11

instance, maternally expressed 3 (MEG3) induces the accumulation of p53 and dramatically increases p53dependent transcription from a p53-responsive promoter [44,45]. LincRNA-p21, which is activated by p53, serves as a key factor in the p53-dependent pathway [46]. Lastly, MALAT1 (metastasis associated lung adenocarcinoma transcript 1) can impact the cell cycle and cellular proliferation by inactivation of p53 and its target genes [26]. Thus, we hypothesized that p53 may also mediate GAS5 function. We found that GAS5 overexpression can activate p53 by posttranscriptional regulation and stimulate expression of its target gene p21. Because the p53-dependent pathway mentioned above can only happen in cells containing wide-type p53, lncRNAs are also involved in a p53-independent pathway exerting different biological effects on p53deficient or -mutant cells, such as H1650. Earlier studies have shown that the interaction between lncRNAs and the transcription factor E2F1 is complicated. E2F1 can be recruited to the imprinted lncRNA H19 promoter, promoting the cell cycle progression of a breast cancer cell [47]. Moreover, MALAT1 is capable of modulating E2F1 activity by regulating Pc2 polycomb protein-mediated sumoylation of E2F1 [48]. Therefore, E2F1 may be another key downstream mediator of GAS5. Indeed, ectopic GAS5 expression in H1650 cells results in the inhibition of E2F1 expression. GAS5 may regulate E2F1 expression via the Pc2 polycomb protein. To confirm this possibility, further study is needed. In this study, we demonstrated for the first time that GAS5 lncRNA has a tumor suppression function mediated by p53-independent and p53-dependent pathways in NSCLC. However, the pathway primarily responsible for the biological function observed with GAS5 overexpression remains to be determined. Here, we report for the first time that GAS5 downregulation is involved in NSCLC tumorigenesis and progression, and GAS5 overexpression can dramatically induce apoptosis and growth arrest in vitro and reduce tumor growth in vivo. Nonetheless, in this study, we just treated NSCLC cells with 5-aza-2-deoxycytidine. Thus, the future studies assessing the changes in the methylation of the CpG islands in the GAS5 promoter regions is needed. In addition, although we found that p53 and E2F1 are key downstream mediators of GAS5, the underlying molecular mechanisms and detailed signaling pathways for GAS5 remain to be clarified. Only with full elucidation of GAS5 functionality and molecular mechanisms relevant to NSCLC can we open avenues for the use of lncRNAs in identification of novel diagnostic or predictive biomarkers and drug targets for NSCLC. ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (No.

12

SHI ET AL.

81170064) and the Natural Science Foundation of China (No. 81302032). We apologize to all researchers whose relevant contributions were not cited due to space limitations.

23. 24.

REFERENCES 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA-Cancer J Clin 2009;59:225–249. 2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CACancer J Clin 2013;63:11–30. 3. Ettinger DS, Akerley W, Bepler G, et al. Non-small cell lung cancer. J Natl Compr Cancer Netw 2010;8:740–801. 4. Koudelakova V, Kneblova M, Trojanec R, Drabek J, Hajduch M. Non-small cell lung cancer - genetic predictors. Biomed Pap Med Fac Univ Palacky Olomouc Czechoslovakia 2013;157: 125–136. 5. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Eng J Med 2008;359:1367–1380. 6. Zhou Q, Zhang XC, Chen ZH, et al. Relative abundance of EGFR mutations predicts benefit from gefitinib treatment for advanced non-small-cell lung cancer. J Clin Oncol 2011;29: 3316–3321. 7. Mattick JS. RNA regulation: A new genetics? Nat Rev Genet 2004;5:316–323. 8. Johnson JL, Pillai S, Chellappan SP. Genetic and biochemical alterations in non-small cell lung cancer. Biochem Res Int 2012;2012:940405. 9. Gibb EA, Brown CJ, Lam WL. The functional role of long noncoding RNA in human carcinomas. Mol Cancer 2011;10:38. 10. Spizzo R, Almeida MI, Colombatti A, Calin GA. Long noncoding RNAs and cancer: A new frontier of translational research? Oncogene 2012;31:4577–4587. 11. van Bakel H, Hughes TR. Establishing legitimacy and function in the new transcriptome. Brief Funct Genomic Proteomic 2009;8:424–436. 12. Wapinski O, Chang HY. Long noncoding RNAs and human disease. Trends Cell Biol 2011;21:354–361. 13. Gutschner T, Diederichs S. The hallmarks of cancer: A long non-coding RNA point of view. RNA Biol 2012;9:703–719. 14. Leygue E, Dotzlaw H, Watson PH, Murphy LC. Expression of the steroid receptor RNA activator in human breast tumors. Cancer Res 1999;59:4190–4193. 15. Hung T, Wang Y, Lin MF, et al. Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters. Nat Genet 2011;43:621–629. 16. Schneider C, King RM, Philipson L. Genes specifically expressed at growth arrest of mammalian cells. Cell 1988;54: 787–793. 17. Coccia EM, Cicala C, Charlesworth A, et al. Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development. Mol Cell Biol 1992; 12:3514–3521. 18. Muller AJ, Chatterjee S, Teresky A, Levine AJ. The gas5 gene is disrupted by a frameshift mutation within its longest open reading frame in several inbred mouse strains and maps to murine chromosome 1. Mamm Genome 1998;9:773–774. 19. Smith CM, Steitz JA. Classification of gas5 as a multi-smallnucleolar-RNA (snoRNA) host gene and a member of the 50 terminal oligopyrimidine gene family reveals common features of snoRNA host genes. Mol Cell Biol 1998;18:6897–6909. 20. Kino T, Hurt DE, Ichijo T, Nader N, Chrousos GP. Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. Sci Signal 2010;3:ra8. 21. Mourtada-Maarabouni M, Hedge VL, Kirkham L, Farzaneh F, Williams GT. Growth arrest in human T-cells is controlled by the non-coding RNA growth-arrest-specific transcript 5 (GAS5). J Cell Sci 2008;121:939–946. 22. Wu W, Bhagat TD, Yang X, et al. Hypomethylation of noncoding DNA regions and overexpression of the long noncoding RNA, AFAP1-AS1, in Barrett's esophagus and Molecular Carcinogenesis

25.

26.

27. 28. 29. 30.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

48.

esophageal adenocarcinoma. Gastroenterology 2013;144: 956–966; e954 Yang F, Huo XS, Yuan SX, et al. Repression of the long noncoding RNA-LET by histone deacetylase 3 contributes to hypoxia-mediated metastasis. Mol Cell 2013;49:1083–1096. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: A link between cancer genetics and chemotherapy. Cell 2002;108: 153–164. Derrien T, Johnson R, Bussotti G, et al. The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression. Genome Res 2012;22: 1775–1789. Tripathi V, Shen Z, Chakraborty A, et al. Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB. PLoS Genet 2013;9:e1003368. Huarte M, Rinn JL. Large non-coding RNAs: Missing links in cancer? Hum Mol Genet 2010;19:R152–161. Perez DS, Hoage TR, Pritchett JR, et al. Long, abundantly expressed non-coding transcripts are altered in cancer. Hum Mol Genet 2008;17:642–655. Garcia-Claver A, Lorente M, Mur P, et al. Gene expression changes associated with erlotinib response in glioma cell lines. Eur J Cancer 2013;49:1641–1653. Mourtada-Maarabouni M, Pickard MR, Hedge VL, Farzaneh F, Williams GT. GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer. Oncogene 2009;28:195–208. Qiao HP, Gao WS, Huo JX, Yang ZS. Long Non-coding RNA GAS5 Functions as a Tumor Suppressor in Renal Cell Carcinoma. Asian Pac J Cancer Prev 2013;14:1077–1082. Tani H, Torimura M, Akimitsu N. The RNA degradation pathway regulates the function of GAS5 a non-coding RNA in mammalian cells. PLoS ONE 2013;8:e55684. Zhang Z, Zhu Z, Watabe K, et al. Negative regulation of lncRNA GAS5 by miR-21. Cell Death Differ 2013;20:1558–1568. Kim H, Kwak NJ, Lee JY, et al. Merlin neutralizes the inhibitory effect of Mdm2 on p53. J Biol Chem 2004;279:7812–7818. Mayo LD, Donner DB. The PTEN, Mdm2, p53 tumor suppressoroncoprotein network. Trends Biochem Sci 2002; 27:462–467. Ouchi T, Monteiro AN, August A, Aaronson SA, Hanafusa H. BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 1998;95:2302–2306. Roe JS, Kim H, Lee SM, Kim ST, Cho EJ, Youn HD. p53 stabilization and transactivation by a von Hippel-Lindau protein. Mol Cell 2006;22:395–405. Sherr CJ, Weber JD. The ARF/p53 pathway. Curr Opin Genet Dev 2000;10:94–99. el-Deiry WS. Regulation of p53 downstream genes. Semin Cancer Biol 1998;8:345–357. Harris SL, Levine AJ. The p53 pathway: Positive and negative feedback loops. Oncogene 2005;24:2899–2908. Vousden KH, Lu X. Live or let die: The cell's response to p53. Nat Rev Cancer 2002;2:594–604. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307–310. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annu Rev Biochem 2012;81:145–166. Zhou Y, Zhang X, Klibanski A. MEG3 noncoding RNA: A tumor suppressor. J Mol Endocrinol 2012;48:R45–R53. Zhou Y, Zhong Y, Wang Y, et al. Activation of p53 by MEG3 non-coding RNA. J Biol Chem 2007;282:24731–24742. Huarte M, Guttman M, Feldser D, et al. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 2010;142:409–419. Berteaux N, Lottin S, Monte D, et al. H19 mRNA-like noncoding RNA promotes breast cancer cell proliferation through positive control by E2F1. J Biol Chem 2005;280: 29625–29636. Yang L, Lin C, Liu W, et al. ncRNA- and Pc2 methylationdependent gene relocation between nuclear structures mediates gene activation programs. Cell 2011;147:773–788.