Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of EBV infected human A549 cells exposed to 10ng/ml TGF beta1 for 4 hours

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease; EBV has previously been localised to alveolar epithelial cells of IPF patients. The molecular process of the epithelial mesenchymal transition (EMT) in IPF remains still unknown. Using an oligonucleotide array analysis, we observed dysregulated expression of members of non-canonical Wnt family in EBV infected A549 after TGF?1 exposure. TGF?1 exposure induced EMT increasing ?-Smooth Muscle Actin (ACTC) and Wnt5b gene expression, but decreasing E-cadherin and DKK1. When data were analyzed as a function of Wnt5b in EMT, significance differences in ACTC and E-cadherin gene expression, active TGF?1 protein levels and collagen deposition could be detected. Treatment with 9-cis Retinoic Acid (9-cisRA) significantly inhibited Wnt5b expression in both EBV infected and non-infected A549, followed by decreased collagen deposition and active TGF?1 protein level. Specific non-canonical Wnt-signalling genes are dysregulated in EBV infected cells and A549 treated with TGF?1; while, 9-cisRA treatment appears to attenuate EMT process in vitro. Experiment Overall Design: EBV infected cells and A549 were cultured in RPMI1640+5%FCS, and exposed to 10ng/ml TGF beta1 for 4hours. RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis were performed as previously reported (Kieran et al., 2003). All analysis were microarrayed in duplicate. Image files were obtained through Affymetrix GeneChip software (MAS5), subsequently robust multichip analysis (RMA) was performed. Expression data were compared to control, p<0.05 correlated values and a signal log ratio of 0.6 or greater (equivalent to a fold change in expression of 1.5 or greater) were taken to identify significant differential regulation (Bolstad et al., 2003). All the SLRs data resulting from the comparative analyses in duplicate were reported in a scatter plot graph to determine the reliability of the assay and the linearity by r2. For all the microarray assays r2 value was higher than 0.98. Using normalised RMA values by Gene Cluster Software, Average Linkage Hierarchical Cluster Analysis was performed using TreeView analysis software (Eisen et al., 1998). Lists of dysregulated genes in both TGF?1 exposed cell lines were curated via the publicly available DAVID, Gene-Ontology (GOCharts) and Functional Annotation Clustering databases (Dennis et al., 2003).

ORGANISM(S): Homo sapiens

SUBMITTER: Andrea Patricelli Malizia

PROVIDER: E-GEOD-5457 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix proteins deposition. Epstein - Barr virus (EBV) has previously been localised to alveolar epithelial cells of IPF patients. In this study we utilised a microarray based differential gene expression analysis strategy to identify potential molecular drivers of EBV associated lung fibrosis. We employed an alveolar epithelial cell line infected with EBV (A-Akata). Lytic phase infection induced in the A-Akata cells by TPA/BA treatment resulted in increase of TGFbeta1 and TIEG1 mRNA expression. Treatment of the A-Akata cells with ganciclovir,; resulted in a down regulation of both TIEG1 and TGFbeta1 expression, accompanied by a suppression of the EBV early response genes, Rta and Zta. This suppression of cell turnover mediators was correlated with an increase in cell activity index. To identify a possible role for Rta in driving apoptotic gene expression, we inhibited the Rta gene expression by silencing RNA, resulting in a decrease in TGFbeta1 and TIEG1 expression. This study identifies an apoptotic role of the EBV early response genes, as enhancer factors of TIEG1 and TGFbeta1 in EBV infected alveolar epithelial cells, potentially providing a possible mechanism for the role of EBV infection in pulmonary fibrosis. Experiment Overall Design: RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis of A549, EBV infected A549 (AAKata) and A549 +TGFbeta1 10ng/ml 4hours were performed as previously reported. All analysis were microarrayed in duplicate. Image files were obtained through Affymetrix GeneChip software (MAS5). Subsequently robust multichip analysis (RMA) was performed. Expression data was compared to control by ANOVA analysis, p<0.05 correlated values and a signal log ratio of 0.6 or greater (equivalent to a fold change in expression of 1.5 or greater) were taken to identify significant differential regulation. All the SLRs data resulting from the comparative analyses were reported in a graph to determine the reliability of the assay and the linearity by r2. For all the microarray assays r2 value was higher than 0.98. Using gene expression values normalised by RMA, Average Linkage Hierarchical Cluster Analysis was performed and the results visualized by TreeView software.

Project description:Epithelial-mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non-small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA) -related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) in NSCLC. MiRNA expression profiles were examined before and after transforming growth factor-beta1 (TGF-M-NM-21) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. MiRNA array and quantitative RT-PCR revealed that TGF-M-NM-21 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN (phosphatase and tensin homolog), was diminished in A549 cells with EMT after the TGF-M-NM-21 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-M-NM-21-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-M-NM-21-induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, whose suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be new therapeutic targets in advanced lung adenocarcinoma patients, depending on the EMT phenomenon. miRNA expression profiles before and after TGF-M-NM-21 exposure were assessed in the four lung adenocarcinoma cell lines, A549, LC2/ad, PC3, and, PC9 by TaqMan miRNA arrays. Relative ratios of miRNAs in cells after TGF-M-NM-21 exposure were calculated when compared with the cells before TGF-M-NM-21 exposure.

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Transcription profiling of EBV infected human A549 cells exposed to 10ng/ml TGF beta1 for 4 hours

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