Gene expression in an EMT model using an immortalized bronchial epithelial cell line induced by TGF-M-NM-21
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Bangrong Cao
PROVIDER: E-GEOD-40374 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Pre-EMT GIF14 cells treated with TGF-M-NM-21 Total RNA obtained from isolated GIF-14 cells from untreated and TGF-M-NM-21-treated sample
2014-02-19 | E-GEOD-44055 | biostudies-arrayexpress
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.
2013-10-30 | E-GEOD-51828 | biostudies-arrayexpress
Project description:Langerhans dendritic cells represent abundantly occuring and evolutionary highly conserved DCs specifically located in the stratified epithelial tissues. LCs are unique among DC family members in that they express epithelial-type adhesion molecules, allowing them to form a tight three-dimensional network in basal and suprabasal epidermal keratinocyte layers and developmentally dependent on the cytokine TGF-M-NM-21. In the present study, we identified BMP-7 as another key factor inducing LC differnetiation. Here we have performed comparative analysis of highly purified CD207+/CD1a+ in vitro generated Langerhans cells in the presence of BMP-7 and TGF-M-NM-21. We have identified that both BMP-7-LCs and TGF-M-NM-21-LCs are closely related to each other. CD34+ hematopoietic stem cells were isolated from freshly obtained cord blood and cultured in the presence of GM-CSF, SCF, Flt3L, TNFa and either TGF-M-NM-21 or BMP-7 for 7 days to get immature LCs. RNA was isolated from these in vitro genetared LCs.
2013-10-18 | E-GEOD-49085 | biostudies-arrayexpress
Project description:Aim: Differentiation of cardiac fibroblasts (Fb) into myofibroblasts (MyoFb) is responsible for connective tissue buildup in myocardial remodeling. We examined reversibility of MyoFb differentiation. Methods and Results: Adult rat cardiac Fb were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different Fb phenotypes. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fiber formation decorated with alpha-smooth muscle actin (M-NM-1-SMA). Transforming growth factor-M-NM-21 (TGF-M-NM-21) promoted terminal differentiation into M-NM-1-SMA positive MyoFb showing near absence of proliferation i.e. non-p-MyoFb (2-fold increase in cell number after 12 days vs 11-fold for p-MyoFb). SD-208, a TGF-M-NM-2-receptor-I kinase blocker, inhibited p-MyoFb differentiation as shown by stress fiber absence, low levels of M-NM-1-SMA protein expression, and high levels of proliferation (32-fold increase after 12 days). Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and 4-fold contraction. Fb produced low levels of collagen and secreted high levels of IL-10. Non-p-MyoFb showed high collagen production and high MCP-1 and TIMP-1 secretion. Transcriptome analysis indicated differential gene expression between all phenotypes. Dedifferentiation of p-MyoFb, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress, shown by stress fiber de-polymerization in 30% of p-MyoFb vs in 8% of non-p-MyoFb. Stress fiber de-polymerization could be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2 day culture in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D culture. Conclusions: Both reduction in mechanical strain and TGF-M-NM-2-receptor-I kinase inhibition can reverse p-MyoFb differentiation but not in non-p-MyoFb. Fibroblasts isolated from each rat heart (n= 4) were cultured in specific conditions to obtain different fibroblast phenotypes: 1) spontaneously differentiation into proliferating myofibroblasts (code RC), 2) terminal differentiation into non-proliferating myofibroblasts with TGF-M-NM-21 (code RT) and 3) inhibition of myofibroblast differentiation with SD-208, a TGF-M-NM-2-receptor-I kinase blocker (code RS). RNA concentration and purity from a total of 12 samples were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit. A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChipM-BM-. Rat Gene 2.0 ST array followed by staining and washing in a GeneChipM-BM-. fluidics station 450 (Affymetrix) according to the manufacturerM-bM-^@M-^Ys procedures. To assess the raw probe signal intensities, chips were scanned using a GeneChipM-BM-. scanner 3000 (Affymetrix).
2014-01-07 | E-GEOD-51824 | biostudies-arrayexpress
Project description:To understand how CK2M-NM-2 silencing promotes a mesenchymal phenotype in human epithelial cells, like TGFM-NM-2 does, we have employed whole genome microarray expression profiling as a discovery platform to compare genes regulated in CK2M-NM-2-depleted cells and in TGFM-NM-2-treated cells. To do so, we began by deriving individual gene expression signatures of WT-, TGFM-NM-2-treated, CK2M-NM-2-depleted or CK2M-NM-2-depleted and rescued with chicken CK2M-NM-2, whose expression is not affected by the shRNA in MCF10A cells, and cataloguing genes exhibiting at least a 1.5-fold up- or down-regulation under each experimental condition. We observed that EMT triggered by TGFM-NM-21 or CK2M-NM-2-depletion induced an overlapping set of changes in gene expression. Of ~25,000 unique genes tested, we found ~1,200 genes whose expression was significantly modulated in TGFM-NM-21-treated or CK2M-NM-2-depleted cells as compared to Mock-cells. Of the 439 genes down-regulated in TGFM-NM-21-treated cells, 74 genes (17%) were also repressed in CK2M-NM-2-depleted MCF10A cells and 45 genes (10%) were commonly up-regulated. As expected, among commonly regulated genes, several mesenchymal genes (CDH2, FN1, MYL9, VIM) were upregulated, whereas epithelial genes such as CDH1, CDH3, CLDN1, CLDN7, OCLN, KRT5, KRT6B, COL2A1 and MUC1, were inversely turned down. The expression of the EMT-inducing transcription factors, Snail and Zeb, was also significantly up-regulated in accordance with the repression of their known target genes. While showing similar phenotypes, the genetic program of TGFM-NM-21-treated or CK2M-NM-2-depleted MCF10A cells also exhibited distinct features. These results highlight that in addition to TGFM-NM-21-regulated genes, CK2M-NM-2-depletion may affect alternative pathways. The microarray analysis was performed with RNAs isolated from parental MCF10A cells and the following MCF10A-derived cells: M-NM-^TCK2M-NM-2- and TGFM-NM-21-treated cells (2ng/ml for 72h) and each from two independent cell cultures (duplicates).
2012-05-06 | E-GEOD-28569 | biostudies-arrayexpress
Project description:Long-term dynamic compression enhanced the mechanical properties of MSC-seeded constructs only when loading was initiated after 21 days of chondrogenic differentiation. This study examined the molecular differences of chondrogenic MSCs compared to undifferentiated MSCs (TGF-beta vs no TGF-beta) and the effects of dynamic loading on MSC chondrogenesis (loading vs free-swelling). Free-swelling MSC-seeded constructs were cultured for 21 days in chemically defined media. Chondrogenesis was induced with TGF-beta3. Undifferentiated controls were maintained in parallel. After 21 days of chondrogenic differentiation, a subset of constructs were subjected to 21 days of dynamic compressive loading. On days 21 and 42, construct mechanical properties and biochemical content were assessed. Microarray analysis was carried out on day 3, day 21 and day 42 constructs. 6 arrays.
2010-11-01 | E-GEOD-18879 | biostudies-arrayexpress
Project description:The role of TGF-M-NM-2-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-M-NM-2 signaling is still unknown. In this study, we observed that the lncRNA-Activated by TGF-M-NM-2 (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB promotes the invasion-metastasis cascade, which suggest that lncRNA-ATB, a mediator of TGF-M-NM-2 signaling, could predispose HCC patients to metastases and may serve as a potential target for anti-metastatic therapies. SMMC-7721 hepatoma cells were continuously treated with 10 ng/ml of recombinant TGF-M-NM-21 for 21 days. Total RNA recovered from three untreated cells and three treated cells were used to acquire different expression profiles of mRNAs and lncRNAs.
2014-02-08 | E-GEOD-54797 | biostudies-arrayexpress
Project description:This study aimed to establish an epithelial-mesenchymal transition (EMT) model with an immortalized human bronchial epithelial cell line, M-BE, and to identify an EMT signature gene set. The TGF-β1-induced M-BE cells got spindle-shaped fibroblast-like morphology and lost the cell-cell contact, with down-regulated expression of epithelial marker E-cadherin and up-regulated expression of mesenchymal markers N-cadherin and Vimentin. Examined by microarray, there were 2628 genes identified as significant EMT-related, including 1490 up-regulated genes (FC > 2, fdr < 0.01) and 1138 down-regulated genes (FC < 0.5, fdr < 0.01) in TGF-β1-induced M-BE cells.
2013-10-06 | GSE40374 | GEO
Project description:Transforming growth factor-M-NM-2 (TGF-M-NM-2) is a key factor for the development of prostate cancer metastases in bone. In breast cancer and melanoma, studies have shown how TGF-M-NM-2 regulates gene expression to allow cancer cells to adapt to the bone microenvironment. We used microarray analyses to characterize the effect of TGF-M-NM-2 on gene expression in prostate cancer cells in vitro. Human prostate cancer cells, PC-3, were cultured in the presence or absence of TGF-M-NM-2. Total RNA was extracted for hybridization on Affymetrix microarray analyzed with Microarray Analysis Suite 5.0.
2014-11-01 | E-GEOD-58698 | biostudies-arrayexpress
Project description:The epithelial to mesenchymal transition (EMT) has been well recognized for many decades as an essential early step in the progression of primary tumors towards metastases. Widespread epigenetic reprogramming of DNA and histone modifications tightly regulates gene expression and cellular activity during carcinogenesis, and epigenetic therapy has been developed to design efficient strategies for cancer treatment. As the first oral agent approved for the clinical treatment of cancer, sorafenib has significant inhibitory effects on tumor growth and EMT. However, a detailed understanding of the underlying epigenetic mechanism remains elusive. In this manuscript, we performed a ChIP-Seq assay to evaluate the activity of sorafenib on the genome-wide profiling of histone modifications. We demonstrate that sorafenib largely reverses the changes in histone modifications that occur during EMT in A549 alveolar epithelial cells. Sorafenib also significantly reduces the coordinated epigenetic switching of critical EMT-associated genes in accordance with their expression levels. Furthermore, we show that sorafenib potentiates histone acetylation by regulating the expression levels of histone-modifying enzymes. Collectively, these findings provide the first evidence that sorafenib inhibits the EMT process through an epigenetic mechanism, which holds enormous promise for identifying novel epigenetic candidate diagnostic markers and drug targets for the treatment of human malignancies. To further explore the underlying epigenetic mechanisms of EMT regulation by sorafenib, we chose conventional markers of active euchromatin such as H3K9ac and H3K4me3, and contrasted their architecture with the repressive structures associated with H3K27me3 and H3K9me3. The profiling of these four selected histone modifications was performed using ChIP-seq on control, TGF-M-NM-21-treated and sorafenib-treated cells. We further performed pair-wise comparisons among the three treatment conditions to assess the changes in the histone modifications within specific genomic regions during EMT.
2012-10-11 | E-GEOD-41479 | biostudies-arrayexpress