GAS5 long non-coding RNA in malignant pleural mesothelioma

Abstract

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA “sponge”. Our aim was to investigate the possible role of the GAS5 in the growth of MPM.MethodsPrimary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry.ResultsGAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression.ConclusionsThe observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.

Lentiviral-mediated overexpression of long non-coding RNA GAS5 reduces invasion by mediating MMP2 expression and activity in human melanoma cells

“…We hypothesize that accumulation of GAS5 in the nucleus may be one of crucial processes of exerting its function. In additon, Renganathan et al revealed that GAS5 is slightly more abundant in the nucleus than that in the cytoplasm in the absence of HhAntag and prevalence in the nucleus was examined after treatment with HhAntag, indicating that HhAntag-induced growth arrest involves accumulation of nuclear GAS5 (18). This study reinforced our hypothesized model.…”

“…GAS5 was originally isolated from NIH 3T3 cells by Schneider et al with the purpose of identifying potential tumor suppressor genes enriched during growth arrest induced by serum starvation (10). GAS5 transcript levels are significantly reduced in a spectrum of cancers including breast, prostate, glioblastoma, renal clear cell, bladder, hepatocellular, pancreatic, non-small cell lung, colorectal, cervical and multiple myeloma (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Importantly, aberrant GAS5 expression was significantly correlated with metastasis in cervical cancer (21) and hepatocellular carcinoma (15).…”

Long non-coding RNA GAS5 inhibits cell proliferation, induces G0/G1 arrest and apoptosis, and functions as a prognostic marker in colorectal cancer

“…The lncRNA GAS5 is non-coding, hosts multiple small nucleolar (sno) RNA sequences in its introns and contains 12 exons (27,28). GAS5 was initially identified during screening for potential tumor suppressor genes (29) and is a stress-inducible gene, which is differentially expressed in healthy and tumor tissues/cell lines (30). In addition, GAS5 has been demonstrated to be involved in the regulation of the cell cycle (31) and to function as a tumor suppressor in human T cells, and breast and prostate cancer cell lines by inducing apoptosis (10,11,32,33).…”

Long non-coding RNA growth arrest-specific transcript 5 in tumor biology

“…However, the putative open reading frame is small and poorly conserved (13,21). GAS5 is non-protein coding but its RNA is spliced, polyadenylated, and associated with ribosomes (22). The essential biological activity of GAS5 is mediated through the introns, which encode multiple snoRNAs (12).…”