Project description:Investigate the difference in gene expression in TGFbeta induced EMT in lung cancer cell line A549 in comparison to uninduced A549 cells.
Project description:Epithelial–mesenchymal transition (EMT) is a plastic process that converts epithelial cells into migratory and invasive cells. Accumulating evidence indicates that EMT is a key event for metastasis in several types of cancer, including non-small cell lung cancer (NSCLC). Especially, transforming growth factor-beta (TGF-beta) acts as a potent inducer of EMT and contributes to cancer progression. Emerging studies suggest that a metabolic reprograming is essential to acquire the EMT phenotype in cancer cells. However, a comprehensive understanding of metabolism in cancer EMT remains largely unexplored. Here, we analyzed metabolic changes during TGF-beta-induced EMT in NSCLC A549 cells using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). At the same time, we examined the expression of metabolic-related genes using microarray analysis.
Project description:Transforming growth factor- (TGF-) signaling is a critical driver of epithelial–mesenchymal transition (EMT) and cancer progression. However, the regulatory roles of long non-coding RNAs (lncRNAs) in TGF--induced EMT and cancer progression are not well understood. Here, we identified an unannotated nuclear lncRNA LETS1 (LncRNA Enforcing TGF- Signaling 1) as a novel TGF-/SMAD target gene. Loss of LETS1 attenuates TGF--induced EMT, migration and extravasation in breast and lung cancer cells. LETS1 potentiates TGF-/SMAD signaling by stabilizing cell surface TGF- type I receptor (TRI) and thereby forms a positive feedback loop. Mechanistically, LETS1 inhibits TRI polyubiquitination by inducing the orphan nuclear receptor 4A1 (NR4A1) expression, a critical determinant of a destruction complex for inhibitory SMAD7. An unbiased interactome analysis identified the Nuclear Factor of Activated T Cells (NFAT5) as a protein partner of LETS1 to mediate activation of NR4A1 promoter. Overall, our findings characterize LETS1 as an EMT-promoting lncRNA and elucidate the mechanism by which nuclear LETS1 potentiates TGF- receptor signaling.
Project description:The goal of this study is to characterize time course gene expression profiles during TGF-beta induced EMT. In particular, we aim to identify and characterize master transcription factors regulate the transition into partial-EMT state. A time series mRNA profile in A549 cells is generated from TGF-beta induced EMT samples during 0h,6h,12h,24h,36h,48h,72h and 96h by deep sequencing, in duplicate, using Illumina HiSeq 2500
Project description:mRNA-seq of A549 cells carrying out EMT-MET in the absence or presence of the EZH2 inhibitor GSK126. In addition, we carried out ChIP-seq of EZH2 in A549 cells upon TGF-B treatment.
Project description:Time Course of TGF-beta treatment of A549 lung adenocarcinoma cell line on Affymetrix HG_U133_plus_2 arrays; triplicate experiments. The goal of the experiment is to profile temporal gene expression changes during TGF-beta-induced epithelial-mesenchymal transition (EMT). During EMT cancer cells loose their epithelial specifc proteins and gain mesenchymal proteins to acquire migratory and invasive phenotype essential for metastasis. Human A549 lung adenocarcinoma cell line was treated with 5 ng/mL TGF-beta for 0, 0.5, 1, 2, 4, 8, 16, 24, and 72 h to induce EMT. The experiment was repeated 3 times. Samples were assayed using Affymetrix HG_U133_plus_2 arrays with 54675 probe-sets, using standard techniques. We provide the raw .CEL files and a supplementary Excel spreadsheet with log-transformed data and selected results from a statistical analysis. Experiment Overall Design: Human A549 lung adenocarcinoma cell line was treated with 5 ng/mL TGF-beta for 0, 0.5, 1, 2, 4, 8, 16, 24, and 72 h. The experiment was repeated 3 times. Samples were assayed using Affymetrix HG_U133_plus_2 arrays with 54675 probe-sets, using standard techniques. The 2 h sample of the third experiment was not run on an array due to poor RNA, so that only 26 arrays were run.
Project description:Time Course of TGF-beta treatment of A549 lung adenocarcinoma cell line on Affymetrix HG_U133_plus_2 arrays; triplicate experiments. The goal of the experiment is to profile temporal gene expression changes during TGF-beta-induced epithelial-mesenchymal transition (EMT). During EMT cancer cells loose their epithelial specifc proteins and gain mesenchymal proteins to acquire migratory and invasive phenotype essential for metastasis. Human A549 lung adenocarcinoma cell line was treated with 5 ng/mL TGF-beta for 0, 0.5, 1, 2, 4, 8, 16, 24, and 72 h to induce EMT. The experiment was repeated 3 times. Samples were assayed using Affymetrix HG_U133_plus_2 arrays with 54675 probe-sets, using standard techniques. We provide the raw .CEL files and a supplementary Excel spreadsheet with log-transformed data and selected results from a statistical analysis.
Project description: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).
Project description:To find out genes regulated by TGF-β in A549 cells, we compared gene expression of cells treated with 1ng/ml TGF-β versus non-treated cells and find out that expression of one transmembrane protein, TM4SF20, is reduced by TGF-β. A549 cells were treated with or without 1ng/ml TGF-β for 12h. RNA was extracted and hybridized on Affymetrix microarrays
Project description:Curcumin (diferuloylmethane) is the bioactive phenolic compound, and the mechanism by which curcumin exerts its anti-metastatic effects was comprehensive and diverse. Several studies reported the anti-metastasis effect of curcumin by its ability to modulate the epithelial-to-mesenchymal transition (EMT) process in different cancers, and the underlying molecular mechanism is poorly understood. EMT is a highly conserved biological process in which epithelial cells acquire mesenchymal-like characteristics by losing their cell-cell junctions and polarity deviating cellular mechanism towards cancer metastasis triggering cancer cells to escape from a primary site to distant locations causing spread of cancer to the entire system ultimately leading to death. In this perspective, we explored the anti-metastatic potential and mechanism of curcumin on the EMT process by establishing in-vitro EMT model using A549 cells induced by TGF-β1. Our results showed that curcumin inhibited EMT by regulating the expression of crucial mesenchymal markers such as MMP2, vimentin, and N-Cadherin. Besides, the transcriptional analysis revealed that curcumin treatment differentially regulated the expression of 75 genes in the NanoString n-counter platform. Further PPI (Protein-protein interaction) network and clusters analysis of differentially expressed genes (DEGs) revealed their involvement in essential biological processes. Altogether, the analysis gives a comprehensive overview of the anti-metastatic effect of curcumin in inhibiting TGF-β1 induced EMT in A549 cells.