LncRNAs have been shown to play a critical role in tumorigenesis and contributes to a diverse of biological functions in human cancers32,33. 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 NSCLC17,34,35. 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 cancer17,28. 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 lines29. 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 EMT36,37, an initial event to promote tumor cell invasion and metastasis30,38. A number of studies have revealed an associative relationship between EMT and lung cancer metastasis39,40. 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 cells41. 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 cells42. Some studies suggest that deregulation of histone acetylation and deacetylation also plays an important role in aberrant expression of lncRNAs, such as lncRNA-LET26. Moreover, lncRNA array analysis show that thousands of lncRNAs are regulated by the pan-HDAC inhibitor LBH589 (panobinostat) in wilms tumor cells43. The data we presented here highlight that GAS5-AS1 can be induced by two pan-HDAC inhibitors, panobinostat and SAHA (Fig. 7C,D). Moreover, specific knockdown of HDAC1 or HDAC3 also enhances expression of GAS5-AS1 in both H1299 and PC-9 cells (Supplementary 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 agents29 and (Supplementary 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.

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.