Downregulation of the long noncoding RNA GAS5-AS1 contributes to tumor metastasis in non-small cell lung cancer

Long noncoding RNA (lncRNA) plays pivotal roles in cancer development. To date, only a small number of lncRNAs have been characterized at functional level. Here, we discovered a novel lncRNA termed GAS5-AS1 as a tumor suppressor in non-small cell lung cancer (NSCLC). The expression of GAS5-AS1 in NSCLC tumors was much lower than that in the adjacent normal lung tissues. The reduced GAS5-AS1 was significantly correlated with larger tumors, higher TNM stages, and lymph node metastasis in NSCLC patients. While ectopic expression or specific knockdown of GAS5-AS1 had no effect on proliferation, cell cycle progression, and apoptosis, it dramatically decreased or increased, respectively, NSCLC cell migration and invasion. Overexpression of GAS5-AS1 in NSCLC cells reduced a cohort of molecules (ZEB1, N-cadherin, Vimentin, and/or Snail1) critical for epithelial-mesenchymal transition (EMT). Furthermore, the DNA demethylating agent 5-aza-2-deoxycytidine failed to upregulate GAS5-AS1 in NSCLC cells, whereas the pan-HDAC inhibitors panobinostat and SAHA significantly induced GAS5-AS1 in a dose-dependent manner. In addition, GAS5-AS1 can be upregulated by specific knockdown of HDAC1 or HDAC3. Collectively, our data suggest that histone modifications play a major role leading to epigenetic silencing of GAS5-AS1 in NSCLC and subsequently promote tumor metastasis via upregulation of several key EMT markers.

which may improve our understanding of the molecular basis of cancer initiation and progression. It has been shown that lncRNAs can function as oncogenes or tumor suppressors in a wide variety of human cancers 17,[18][19][20] , including NSCLC 21 . However, the underlying mechanisms of lncRNA deregulation remain elusive. Methylation in the promoter region of lncRNAs has been found to result in aberrant expression of lncRNAs in human cancers [22][23][24] . Histone acetylation in the promoter region can also affect lncRNA transcriptional activation 25,26 . Thus, it indicated that epigenetic regulatory factors, including histone acetylation or DNA methylation, could manipulate the expression of lncRNAs.
Recent studies reveal that lncRNAs play a critical role in NSCLC pathogenesis 27,28 , providing a new avenue to explore the biology of this disease. We previously showed that the lncRNA GAS5 (growth arrest-specific transcript 5) was significantly downregulated in NSCLC tissues and cell lines; and elevated expression of GAS5 inhibited cell proliferation and induced apoptosis in NSCLC cells 29 . Nonetheless, as the antisense RNA of GAS5, GAS5-AS1's expression and its biological role in NSCLC development and progression remains unknown. Here, we discovered that the reduced expression of GAS5-AS1 in NSCLC samples as compared to the adjacent normal lung tissues was significantly correlated with TNM stages, tumor size, and lymph node metastasis. We have also investigated the role of GAS5-AS1 in regulating NSCLC cell migration and invasion and explored the potential mechanism leading to downregulation of GAS5-AS1 in NSCLC.

Results
The expression of GAS5-AS1 is significantly downregulated in NSCLC cells. To determine whether the lncRNA GAS5-AS1 plays a role in the development of NSCLC, we first performed qRT-PCR assays to measure the expression levels of GAS5-AS1 in 48 NSCLC tumors and their self-paired adjacent normal lung tissues. The expression of GAS5-AS1 was decreased in majority of the NSCLC tumors. Statistical analysis indicated that GAS5-AS1 was significantly downregulated in NSCLC tumors as compared to the normal tissues (Fig. 1A). Moreover, the reduced expression of GAS5-AS1 in NSCLC was associated with larger tumor size (> 3 cm, P = 0.007), higher TNM stage (P = 0.012), and lymph node metastasis (P = 0.018) (Fig. 1B). However, the expression of GAS5-AS1 had no significant correlation with other parameters, such as age, gender, differentiation, smoking history, and histology type in NSCLC (Table 1). We next examined this lncRNA's expression in a number of NSCLC cell lines, and discovered a much lower expression of GAS5-AS1 in 4 out of six NSCLC cell lines as compared to human bronchial epithelial (HBE) cells ( Fig. 2A). Thus, significant downregulation of GAS5-AS1 is frequently observed in majority of the NSCLC tumors and cell lines.

Ectopic expression or downregulation of GAS5-AS1 influences NSCLC cell migration and invasion without altering cell proliferation and apoptosis in vitro.
To assess the biological function of GAS5-AS1 in NSCLC, we first examined the impact of GAS5-AS1 expression on the proliferation and apoptosis of NSCLC cells. Overexpression of GAS5-AS1 was achieved via transient transfection of pCDNA-GAS5-AS1 into H1299 or PC-9 cells. The expression of GAS5-AS1 was significantly increased as compared to the same cells transfected with empty vector (Fig. 2B). However, ectopic expression of GAS5-AS1 did not impair the growth of H1299 and PC-9 cells as compared to the empty vector-transfected cells (Fig. 3A). Cell cycle analysis of the H1299 or PC-9 cells transfected with pCDNA-GAS5-AS1 or empty vector showed no significant alterations in the percentage cells of G1/G0, S, and G2/M phases (Fig. 3B). Additionally, we performed flow cytometric analysis of apoptotic cells with Annexin V staining, and found that the increased GAS5-AS1 expression in H1299 or PC-9 cells did not induce apoptosis (Fig. 3C). To further assess the importance of GAS5-AS1 in NSCLC progression, we wondered whether GAS5-AS1 expression might alter the metastatic potential of NSCLC cells. We thus performed Boyden chamber transwell assays to examine cell migration and invasion. The increased GAS5-AS1 expression significantly impeded the migration of H1299 and PC-9 cells. Similarly, the invasiveness of H1299 and PC-9 cells-transfected with pCDNA-GAS5-AS1 was also dramatically reduced ( Fig. 4), indicating that ectopic expression of GAS5-AS1 inhibited migratory and invasive phenotype of NSCLC cells. Conversely, we performed reciprocal experiments to examine the effects of GAS5-AS1 knockdown on cell proliferation and apoptosis as well as migration and invasion in SPC-A1 cells, which express high levels of GAS5-AS1 ( Fig. 2A). Significant downregulation of GAS5-AS1 in SPC-A1 cells upon specific siRNA transfection was confirmed by qRT-PCR analysis (Fig. 5A). Compared to the negative control siRNA-transfected cells, specific knockdown of GAS5-AS1 did not change cell proliferation (Fig. 5B), cell cycle progression (Fig. 5C), and apoptosis (Fig. 5D). In contrast, specific knockdown of GAS5-AS1 in SPC-A1 cells significantly increased cell migration and invasion (Fig. 5E,F). Taken together, our data demonstrate that altered expression of GAS5-AS1 does not affect NSCLC cell proliferation and apoptosis in vitro. The biological function of GAS5-AS1 in NSCLC may influence tumor metastasis via regulating cell migration and invasion.
Elevated expression of GAS5-AS1 seems to inhibit the expression of several EMT markers in NSCLC cells. Since epithelial-mesenchymal transition (EMT) is a key step in the process of tumor infiltration or progression, and it is a critical mechanism underlying metastasis and cancer cell invasion 30,31 . We next sought to explore whether there was any interaction between EMT-related markers and GAS5-AS1 expression. The expression levels of GAS5-AS1 in H1299 or PC-9 cells gradually increased via transfection with different concentrations of pcDNA-GAS5-AS1 (Fig. 6A,B). Overexpression of GAS5-AS1 in H1299 cells reduced ZEB1, N-cadherin, and Vimentin in a dose-dependent manner (Fig. 6C), whereas the expression of ZEB1, N-cadherin, and Snail1 protein was gradually decreased upon ectopic expression of GAS5-AS1 in PC-9 cells (Fig. 6D). Collectively, our data suggest that GAS5-AS1 modulates the metastatic potential of NSCLC cells in part through its influence on expression of several key EMT markers.

HDAC inhibitors induce expression of GAS5-AS1 in NSCLC cells.
We previously showed that promoter methylation seemed to contribute to the reduction of GAS5-AS1's sister lncRNA GAS5 in NSCLC 29 . Indeed, bioinformatics analysis identified CpG islands in the GAS5 promoter region (Fig. 7A). However, no CpG island was found in the promoter region of GAS5-AS1 (Fig. 7B), suggesting that DNA methylation might not be the major mechanism resulting in downregulation of GAS5-AS1 in NSCLC. To verify our prediction, we treated H1299 and PC-9 cells with the demethylating agent decitabine at different concentrations. As expected, while decitabine was able to induce expression of GAS5, it had no significant effect on GAS5-AS1 expression (Supplementary Figure S1). Histone modification is another important mechanism controlling transcriptional activation of lncRNAs. Hence, we next investigated whether the aberrant expression of GAS5-AS1 in NSCLC was attributed to the changes of histone acetylation. H1299 and PC-9 cells were treated with the pan inhibitors of histone deacetylase (HDAC), SAHA or panobinostat. We discovered that the expression levels of GAS5-AS1 were significantly upregulated by both SAHA and panobinostat in a dose-dependent manner (Fig. 7C,D). To provide a direct evidence supporting the impact of histone modification on GAS5-AS1 expression in NSCLC cells, we performed additional experiments and found that specific knockdown of HDAC1 or HDAC3 with siRNA was able to significantly enhance the expression levels of GAS5-AS1 in H1299 (Supplementary Figure S2A) and PC-9 cells (Supplementary Figure S2B). Taken together, our data indicate that histone deacetylation, but not promoter methylation at least partially contributes to the downregulation of GAS5-AS1 in NSCLC. The expression of GAS5-AS1 was significantly lower in larger tumors. The expression of GAS5-AS1 was significantly lower in the NSCLC patients with an advanced TNM stage than those with an early TNM stage, and was also significantly lower in the NSCLC patients with lymph node metastasis than those with no lymph node metastasis.

Discussion
LncRNAs have been shown to play a critical role in tumorigenesis, and contributes to a diverse of biological functions in human cancers 32,33 . To date, the roles of vast majority of lncRNAs in NSCLC initiation and progression are far from being fully elucidated. Tumor metastasis is the major cause of death in patients with NSCLC, thus understanding the molecular mechanism by which a specific lncRNA is involved in metastasis may provide novel opportunities to identify effective therapy against NSCLC. Recent studies suggest that several lncRNAs are dysregulated in multiple cancers, including NSCLC 17,34,35 . One of these is metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched abundant transcript 2 (NEAT2), which is a highly conserved nuclear lncRNA and a predictive marker for metastasis in lung cancer 17,28 . In the current study, we discovered that the lncRNA GAS5-AS1 was downregulated in NSCLC tumors as compared to the adjacent normal lung tissues, and the expression of GAS5-AS1 was significantly lower in NSCLC patients with later stages of tumors and lymph node metastasis. These findings indicate that GAS5-AS1 may function as a tumor suppressor in the modulation of NSCLC progression.
As the antisense RNA of GAS5, GAS5-AS1 (NCBI no. NR_037605.1) is a novel lncRNA transcript which maps on chromosome 1. Our recent studies revealed that GAS5 was also significantly downregulated in NSCLC tumors and cell lines 29 . While GAS5 was found to mainly inhibit proliferation and induce apoptosis in NSCLC cells, the role of GAS5-AS1 in NSCLC remained unknown. We thus investigated the effects of GAS5-AS1 overexpression on various biology aspects of NSCLC cells. Two NSCLC cell lines (H1299 and PC-9) that are commonly used and express low levels of GAS5-AS1 ( Fig. 2A) were chosen to evaluate the effect of GAS5-AS1 on cell behavior. We observed that elevated expression of GAS5-AS1 did not alter cell proliferation and cell cycle progression, and had no influence on the cells undergoing apoptosis in the NSCLC cells tested (Fig. 3). Instead, ectopic expression of GAS5-AS1 led to a dramatic inhibition of migration and invasion in both H1299 and PC-9 cells (Fig. 4). Conversely, similar conclusions were also supported by the reciprocal experiments. Specific knockdown of GAS5-AS1 expression in SPC-A1 cells, which express high levels of GAS5-AS1 ( Fig. 2A), significantly increased cell migration and invasion without affecting proliferation, cell cycle progression, and apoptosis (Fig. 5). Collectively, our data suggest that while both GAS5 and GAS5-AS1 play tumor suppressive roles in NSCLC development, these two lncRNAs function in a distinct way. i.e. GAS5 mainly inhibits NSCLC cell proliferation and induces apoptosis, whereas GAS5-AS1 is crucial to repress NSCLC cell migration and invasion. These novel findings inspire us to hypothesize that the NSCLC cells with low expression of both GAS5 and GAS5-AS1 may be more aggressive than those with low expression of either one of them. To test this hypothesis, further analyses of additional clinical samples of NSCLC patients are needed. Meanwhile, we are not sure whether co-transfection of GAS5 and GAS5-AS1 into the same NSCLC cell line may induce severe growth inhibition and/or apoptosis. We are currently performing the experiments in our laboratory. To better understand the molecular basis responsible for the inhibition of migration and invasion mediated by GAS5-AS1 in NSCLC, we explored potential targets associated with cell migration and invasion. Elevated expression of GAS5-AS1 in H1299 and PC-9 cells clearly decreased ZEB1 and N-cadherin protein levels in a dose-dependent manner (Fig. 6C,D). The increased GAS5-AS1 also reduced vimentin in H1299 cells and decreased SNAIL1 in PC-9 cells. All of these are well-known markers of EMT 36,37 , an initial event to promote tumor cell invasion and metastasis 30,38 . A number of studies have revealed an associative relationship between EMT and lung cancer metastasis 39,40 . To the best of our knowledge, we are the first providing experimental data showing that GAS5-AS1 contributes to NSCLC cell migration and invasion at least partially through regulation of EMT. Nonetheless, detailed studies of the signaling pathway responsible for the biological functions of GAS5-AS1 in EMT are needed.
It has been reported that tumor suppressor genes are usually inactivated by genetic or epigenetic alterations in cancer cells 41 . The expression of ncRNAs can be modified by epigenetic factors, including DNA methylation and histone modification. A recent study of genome-wide DNA methylome analysis reveals that aberrant promoter methylation contributes to dysregulated ncRNAs in human breast cancer cells 42 . Some studies suggest that deregulation of histone acetylation and deacetylation also plays an important role in aberrant expression of lncRNAs, such as lncRNA-LET 26 . Moreover, lncRNA array analysis show that thousands of lncRNAs are regulated by the pan-HDAC inhibitor LBH589 (panobinostat) in wilms tumor cells 43 . The data we presented here highlight that GAS5-AS1 can be induced by two pan-HDAC inhibitors, panobinostat and SAHA (Fig. 7C,D). Moreover,   Figure S2), suggesting that histone deacetylation may directly involve in regulation of GAS5-AS1 transcriptional activation. Interestingly, a CpG island was found in the promotor region of GAS5 (Fig. 7A), which could be upregulated by DNA demethylating agents 29 Figure S1). In contrast, there is no CpG island in the promotor region of GAS5-AS1 (Fig. 7B) and the DNA demethylating agent does not induce GAS5-AS1 expression in NSCLC cells (Supplementary Figure S1). Taken together, these data further our understanding of the epigenetic role in regulating lncRNA expression in NSCLC.

and (Supplementary
In summary, we discovered that GAS5-AS1 was downregulated in NSCLC tumors and the reduced expression of GAS5-AS1 significantly associated with larger tumors (> 3 cm), higher TNM stage, and lymph node metastasis, Further studies showed that ectopic expression of GAS5-AS1 inhibited NSCLC cell migration and invasion partially through repression of several key EMT markers. It appeared that both GAS5-AS1 and its "sister" lncRNA GAS5 functioned as tumor suppressors in NSCLC, however, they acted via distinct mechanisms. GAS5 mainly inhibits NSCLC cell proliferation and induces apoptosis, whereas GAS5-AS1 is vital to repress NSCLC cell migration and invasion without altering proliferation/survival.

Methods
Patients and tissue samples. Forty eight paired NSCLC and the adjacent non-tumor lung tissues were obtained from patients who underwent surgery at Department of Thoracic Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China, between November 2011 and September 2014. All patients were diagnosed with NSCLC according to histopathological features. The patients' clinicopathological characteristics, including tumor-node-metastasis (TNM) staging, were summarized in Table 1. No preoperative adjuvant therapy was conducted. Following surgery, all the tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C until required. The clinical samples were acquired with written informed consent from all of the participants following protocols approved by the Institutional Review Board of Nanjing University. All experimental procedures used in the study were carried out in accordance with the approved guidelines by the Institutional Review Board of Nanjing University.
Cells and cell culture. Human NSCLC cell lines (A549, SPCA-1, H1299, H1703, H520, and PC-9) and the 16-HBE (HBE bronchial epithelial cells) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). A549, H1703, H520, and HBE cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). SPCA-1, H1299, and PC-9 cells were maintained in DMEM/F12 medium containing 10% FBS. All cell lines were free of mycoplasma contamination, which was determined by the MycoAlert ™ Mycoplasma Detection Kit (Lonza Group Ltd. Basel, Switzerland) The other half of the cells were subjected to western blot analyses with specific antibody. The expression levels of ZEB1, N-cadherin, and Vimentin were determined in H1299 cells, whereas ZEB1, N-cadherin, and SNAIL1 were analyzed in PC-9 cells. every three months. All cell lines were cultured in a 37 °C humidified atmosphere containing 95% air and 5% CO 2 and were split twice a week.