Decreased expression of long noncoding RNA GAS5 indicates a poor prognosis and promotes cell proliferation in gastric cancer

Gastric cancer is the second leading cause of cancer death, and is the most common gastrointestinal malignancy in East Asia, Eastern Europe, and parts of Central and South America [1]. Although the majority of the patients at an early stage of gastric carcinoma can be cured by surgery, more than half of those at an advanced stage of the disease die of carcinoma recurrence, even after undergoing curative gastrectomy [2]. Therefore, better understanding of the pathogenesis and identification of the molecular alterations is essential for the development of useful indicators that aid novel effective therapies for gastric cancer [3–5].

It is well known that protein-coding genes account for only 2% of the total genome, whereas the vast majority of the human genome can be transcripted into noncoding RNAs [6–9]. Among them are long noncoding RNAs (lncRNAs), which are more than 200 nt in length with limited or no protein-coding capacity. LncRNAs are often expressed in a disease-, tissue- or developmental stage-specific manner making these molecules attractive therapeutic targets and pointing toward specific functions for lncRNAs in development and diseases, in particular human cancer [10–13]. Multiple lines of evidence have revealed the contribution of lncRNAs as having oncogenic and tumor suppressor roles in tumorigenesis. A famous oncogenic lncRNA involved in tumor pathogenesis is known as HOTAIR (Hox transcript antisense intergenic RNA), which has been consistently upregulated and identified as a strong prognosis marker of patient outcomes such as metastasis and patient survival in diverse human cancers. The studies also revealed that HOTAIR exerts its oncogenic functions via binding the PRC2 (polycomb repressive complex 2), which methylates histone H3 on K27 to promote gene repression [14–16]. A similar mode of action is executed by the lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), a novel tumor suppressor interacting with the PRC2 complex to block the activity of p15 ^INK4B , a well-known tumor suppressor gene. Moreover, the depletion of ANRIL increases the expression of p15 ^INK4B and inhibits cellular proliferation tumorigenesis [17]. Maternally expressed gene 3 (meg3) also represents a tumor suppressor gene that encodes a MEG3 lncRNA, which expression is lost in an expanding list of primary human tumors, and re-expression of MEG3 could induce cell growth arrest and promote cell apoptosis partly via the activation of P53 [18]. Nevertheless, the overall pathophysiological contributions of lncRNAs to gastric cancer remain largely unknown.

In our current study, which seeks to determine the clinical significance and functions of dysregulated lncRNAs in gastric carcinogenesis, we investigated lncRNA GAS5 (Growth Arrest-Specific Transcript 5), which was previously shown to be consistently downregulated and identified as a tumor-suppressor lncRNA in prostate cancer cells, renal cell carcinoma cells and breast cancer cells [19–21], though its functional significance has not yet been established. In this study, we demonstrated that decreased GAS5 expression was a characteristic molecular change in gastric cancer and investigated the effect of altered GAS5 level on the phenotypes of gastric cancer cells in vitro and in vivo. Then, we analyzed the potential relationship between this lncRNA level in tumor tissues and existing clinicopathological features of gastric cancer, as well as clinical outcome. Our findings suggest that lncRNA GAS5 may represent a novel indicator of poor prognosis in gastric cancer and may be a potential therapeutic target for diagnosis and gene therapy.

LncRNAs dysregulation may affect epigenetic information and provide a cellular growth advantage, resulting in progressive and uncontrolled tumor growth [14–18]. Effective control of both cell survival and cell proliferation is critical to the prevention of oncogenesis and to successful cancer therapy. Therefore, identification of cancer-associated lncRNAs and investigation of their clinical significance and functions may provide a missing piece of the well-known oncogenic and tumor suppressor network puzzle.

GAS5 is a long ncRNA (~650 bases in humans) that was originally isolated from a screen for potential tumor suppressor genes expressed at high levels during growth arrest [23]. Its encoding gene, gas5, comprises 12 exons and encodes ten box C/D snoRNAs within its introns [24]. Two mature GAS5 lncRNAs, GAS5a and GAS5b, have also been identified in humans due to the presence of alternative 5’-splice donor sites in exon 7, whereas GAS5b is the major isoform (NR_002578.2, 77 nt, simply called GAS5 in this study), and GAS5a has only 45 nt, missing 32 nt at the 3’ end [25]. GAS5 has been shown to be aberrantly expressed in prostate cancer, renal cell carcinoma, breast cancer, head and neck squamous cell carcinoma (HNSCC), and glioblastoma multiforme [19–21, 26]. For breast cancer and HNSCC, low GAS5 expression is an adverse prognostic factor for survival. Moreover, overexpression of GAS5 attributed to growth arrest of several cancer cell lines through regulation of apoptosis and cell cycle, under basal conditions or various cell death stimuli, including chemotherapeutic agents, suggesting its clinical significance in the development and therapy of cancer [19–21]. These data demonstrate the potential tumor-suppressor role of GAS5; however, the relationship between expression of GAS5 and gastric cancer development and/or progression remains unclear.

Our studies were designed to investigate the expression and prognostic significance of GAS5 in patients with gastric cancer. GAS5 expression was retrospectively analyzed in 89 patients with gastric carcinoma. Results were assessed for association with clinical features and DFS/OS of gastric cancer patients after gastrectomy. Prognostic values of GAS5 expression and clinical outcomes were also evaluated by Cox regression analysis. The results showed that GAS5 expression was significantly decreased in gastric cancer tissues and cell lines. A lower expression of GAS5 was detected in tumor of larger size, higher tumor stage, deeper depth of invasion and more regional lymph nodes. In addition, the downregulation expression of GAS5 was associated with poor prognosis. Moreover, ectopic expression of GAS5 was demonstrated to decrease gastric cancer cell proliferation and induce apoptosis, while downregulation of endogenous GAS5 could promote cell proliferation in vitro and in vivo. Taken together, these findings indicate that GAS5 could function as a tumor suppressor via regulating cell growth and apoptosis, and may be useful in the development of novel prognostic or progression markers for gastric cancer.

Although GAS5 has been suggested to have a tumor-suppressive role, the underlying mechanism of GAS5-mediated gene expression having an impact on tumorigenesis is still elusive. Kino et al. have found that GAS5 could structurally mimic the glucocorticoid receptor response element (GRE) to suppress GR-induced transcriptional activity of endogenous glucocorticoid- responsive genes [25]. Zhang et al. have provided a possible mechanism for GAS5 as a tumor suppressor, which may be attributed to its ability to suppress the oncogenic miR-21 in breast cancer [27]. Nevertheless, since it’s highly possible that target genes of lncRNAs differ between specific tissues and cell types, specific target genes controlled by GAS5 for gastric pathogenesis remain unknown and deserve investigation. In this study, to explore the molecular mechanism by which GAS5 contributes to cell proliferation of gastric cancer, we investigated potential targets which were responsible for cell cycle arrest and cell growth inhibition. Our present experimental results confirmed that E2F1, as well as Cyclin D1, were functional targets of GAS5 in gastric cells. E2F1 expression has been found to be upregulated in mutiple cancers, and its overexpression contributes to many tumors development by acting as an important transcript factor regulating key regulator genes that controlling cell proliferation [28, 29]. Cyclin D1 is one of the most important proteins to regulate cell cycle, and related with the development of many cancers. Cyclin D1 binds and activates CDK4/6, which subsequently phosphorylates tumor suppressor protein Rb and allows the cell cycle to progress through G1 into S [30]. Furthermore, P21 expression has been shown to be reduced or lost in a variety of cancer types [31]. A possible explanation is that P21 exerts its inhibitory control over the cell cycle primarily through direct binding to cyclins and CDKs, therefore preventing cell proliferation [32]. Here, we also found P21 was a downstream regulator involved in GAS5-mediated growth arrest in gastric cancer cells. Taken together, these findings indicate that lncRNA GAS5 may function as a tumor suppressor and its deficiency or decreased expression could contribute to gastric cancer development; however, further studies are required to clarify GAS5 regulation of the above targets expression in gastric cancer cells.

In summary, we demonstrate that the decreased GAS5 expression is a common event underlying gastric cancer, indicating that GAS5 may play a key tumor-suppressive as an indicator of poor survival rate and a negative prognostic factor for gastric cancer patients. Further well understanding of the mechanisms of GAS5 in the molecular etiology of gastric cancer will promote the development of lncRNA-directed diagnostic and therapeutic agents against this deadly disease.

Ming Sun, Fei-yan Jin, and Rui Xia are joint first authors.