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.
Highlights
Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival
GAS5 Long non-coding RNAs (lncRNA) expression level is lower in MPM cell lines compared to normal mesothelial cells and it is increased by drugs inducing growth arrest GAS5 expression in MPM cell lines (n = 22) is significantly lower (Figure 1A; p < 0.005; individual MPM cell line profile is shown Additional file 1: Figure S1) when compared to normal mesothelial cells (n = 7)
To investigate whether GAS5 expression in MPM cells could be modulated by drugs inducing growth arrest, we treated MPM cells with either HhAntag or with NVPBEZ235 as previously described [25,26]
Summary
Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Molecular studies identified altered expression of critical described and include chromatin modifier, transcriptional and post-transcriptional regulator of gene expression [9,10,11]. In this context, lncRNAs are emerging as mammalian transcription key regulators in response to developmental or environmental signals [12,13,14] and are associated to many cancer related pathways through gene regulation [15]. A minimal region of deletion was identified in asbestos induced murine malignant mesothelioma which includes gas locus [17]. We investigate whether GAS5 has a role in MPM biology
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
