Dataset Information

Long non coding RNAs (lncRNAs) are dysregulated in Malignant Pleural Mesothelioma (MPM).

ABSTRACT: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer that is often diagnosed at an advanced stage and is characterized by a long latency period (20-40 years between initial exposure and diagnosis) and prior exposure to asbestos. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers and despite minor improvements in treatment, median survival rates do not exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) play an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. Here we used NCode long noncoding microarrays to identify differentially expressed lncRNAs potentially involved in MPM pathogenesis. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological significance (>3-fold difference). Expression levels of 9 candidate lncRNAs were technically validated using RT-qPCR, and biologically validated in three independent test sets: (1) 57 archived MPM tissues obtained from extrapleural pneumonectomy patients, (2) 15 cryopreserved MPM and 3 benign pleura, and (3) an extended panel of 10 MPM cell lines. RT-qPCR analysis demonstrated consistent up-regulation of these lncRNAs in independent datasets. ROC curve analysis showed that two candidates were able to separate benign pleura and MPM with high sensitivity and specificity, and were associated with nodal metastases and survival following induction chemotherapy. These results suggest that lncRNAs have potential to serve as biomarkers in MPM.

SUBMITTER: Wright CM

PROVIDER: S-EPMC3747266 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Long non coding RNAs (lncRNAs) are dysregulated in Malignant Pleural Mesothelioma (MPM).

Wright Casey M CM Kirschner Michaela B MB Cheng Yuen Yee YY O'Byrne Kenneth J KJ Gray Steven G SG Schelch Karin K Hoda Mir Alireza MA Klebe Sonja S McCaughan Brian B van Zandwijk Nico N Reid Glen G

PloS one 20130819 8

Malignant Pleural Mesothelioma (MPM) is an aggressive cancer that is often diagnosed at an advanced stage and is characterized by a long latency period (20-40 years between initial exposure and diagnosis) and prior exposure to asbestos. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers and despite minor improvements in treatment, median survival rates do not exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (l ...[more]

PMID: 23976967

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Project description:Malignant Pleural Mesothelioma (MPM) is an aggressive cancer that is often diagnosed at an advanced stage and is characterized by a long latency period (20-40 years between initial exposure and diagnosis) and prior exposure to asbestos. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates do not exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) play an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. Here we used NCode long noncoding microarrays to identify differentially expressed lncRNAs potentially involved in MPM pathogenesis. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological significance (>3-fold difference). Expression levels of 9 candidate lncRNAs were technically validated using RT-qPCR, and biologically validated in three independent test sets: (1) 57 archived MPM tissues obtained from extrapleural pneumonectomy patients, (2) 15 cryopreserved MPM and 3 benign pleura, and (3) an extended panel of 10 MPM cell lines. RT-qPCR analysis demonstrated consistent up-regulation of these lncRNAs in independent datasets. ROC curve analysis showed that two candidates were able to separate benign pleura and MPM with high sensitivity and specificity, and were associated with nodal metastases and survival following induction chemotherapy. These results suggest that lncRNAs have potential to serve as biomarkers in MPM. To identify mRNA and lncRNA biomarkers associated with malignant pleural mesothelioma (MPM), we performed gene expression array analysis on 4 MPM cell lines (H28, MM05, MSTO-211H, H226) compared to the immortalised mesothelial line (MeT-5A). All cell lines were profiled in duplicate. Synthesis of the labelled first strand cDNA was conducted using the Superscript Plus Direct cDNA labeling system with starting material of 10ug total RNA. The labeled dNTP mix was added to the reaction to generate labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA was purified using low elution volume spin cartridges included in the purification module. Samples were labelled using Alexa Fluor 555 dyes. Samples were then hybridised to NCode Long Noncoding RNA Microarrays. Slides were scanned using an Agilent Scanner. Genes differentially expressed between Met-5A and the MPM cell lines were identified on the basis of P-value and fold change.

Project description: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.

Dataset Information

Long non coding RNAs (lncRNAs) are dysregulated in Malignant Pleural Mesothelioma (MPM).

Publications

Long non coding RNAs (lncRNAs) are dysregulated in Malignant Pleural Mesothelioma (MPM).

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