Analysis of gene expression changes during long-term induction of EMT in EpRas tumor cells by TGF-b1.
ABSTRACT: EMT is a developmental process, which can be reactivated in cancer cells. Cancer cells maintaining EMT in the circulation may have pro-metastatic properties. We used microarrays to profile EMT-related gene expression changes after 14 d of TGF-b1 treatment. EpRas cells were cultured in standard conditions either in the presence of absence of recombinant TGF-b1 (2 ng/ml) for 14 d. After this period, RNA was isolated from triplicate wells of treated and non-treated cells, and hybridized on Affymetrix microarrays.
Project description:Alu SINEs are the most numerous frequently occurring transcription units in our genome and possess sequence competence for transcription by RNA Pol III. However, through poorly understood mechanisms, the Alu RNA levels are maintained at very low levels in normal somatic cells with obvious benefits of low rates of Alu retrotransposition and energy-economical deployment of RNA Pol III to the tRNA genes which share promoter structure and polymerase requirements with Alu SINEs. Using comparative ChIP sequencing, we unveil that a repeat binding protein, CGGBP1, binds to the transcriptional regulatory regions of Alu SINEs thereby impeding Alu transcription by inhibiting RNA Pol III recruitment. We show that this Alu-silencing depends on growth factor stimulation of cells and subsequent tyrosine phosphorylation of CGGBP1. Importantly, CGGBP1 ensures a sequence-specific discriminative inhibition of RNA Pol III activity at Alu promoters, while sparing the structurally similar tRNA promoters. Our data suggest that CGGBP1 contributes to growth-related transcription by preventing the hijacking of RNA Pol III by Alu SINEs. This study was used to find out the effect of CGGBP1 on serum-induced changes in gene expression and effect of serum on gene expression regulation by CGGBP1. Gene expression profiling of normal human fibroblasts under 4 different experimental perturbations: serum starvation or serum stimulation and CGGBP1 depletion or normal CGGBP1 levels.
Project description:In the intestine, Hedgehog (Hh) signalling orchestrates epithelial homeostasis in a bidirectional loop. Differentiated enterocytes secrete the ligand leading to active downstream signaling exclusively in the stroma. In turn, Hh-driven stromal factors contribute to the control of intestinal stem cell numbers and induce epithelial differentiation. To investigate the consequences of stromal Hh activation on the gene expression level, we performed microarray analysis on full-thickness colonic tissue from Col1a2CreER;Ptch1fl/fl mice (C57BL/6 background). Upon Cre-driven recombination, these mice lack the inhibiting Hh receptor Ptch1 specifically in Col1a2 expressing stroma cells, leading to stroma-specific activation of downstream Hh signaling. RNA was isolated from 1 cm of fresh-frozen whole colonic tissue from Col1a2CreER;Ptch1fl/fl mice (n = 4) and controls (n = 4) starting 1.5 cm from the anus 7 days after administration of 5mg Tamoxifen i.p. to activate Cre. RNA quality was assessed using the Agilent 2200 Tape Station system; RNA integrity numbers (RIN) >8 were considered sufficient. Affymetrix Mouse Gene ST 2.0 arrays were used. Robust multi-array average (RMA) median polish procedures were applied for normalization.
Project description:Gene expression analysis of BJhTERT RhoA-KO and PC3 mRFP was performed before and after six days confrontation in co-cultivation. Analyses of original and control fibroblasts was also performed. To identify molecules that may mediate the tumor promoting effect of RhoA knock out fibroblasts, we performed gene expression analysis of BJhTERT RhoA-KO and PC3 mRFP cells before the confrontation and then after six days after it, using Affymetrix Whole Transcript Assay platform. Four days old fibroblast monolayer was confronted with PC3 mRFP tumor cells, plated in ratio 1:30 to number of plated fibroblasts. After six days of confrontation cells were sorted by FACS (Fluorescence-activated cell sorting). Total RNA was isolated from each confronted and non-confronted cells with kit. Then 150 ng of total RNA were used for transcriptomic analysis.for RNA extraction and hybridization on Affymetrix microarrays
Project description:The aim of this study was to investigate if milk fat globule membrane (MFGM) enclosing the dairy fat influence peripheral blood mononuclear cells (PBMC) gene expression. This study was a 8-week single-blind, randomized, controlled isocaloric trial with two parallel groups including overweight (mean BMI: 28) adult women (n=30). All subjects consumed 40 g dairy fat per day either as cream (MFGM diet) or as butter oil (control diet). MFGM diet and control diet showed differential responses in plasma cholesterol. Absolute differences in gene expression were calculated for each gene in each subject, comparing after with before the intervention. These absolute differences in gene expression were compared between intervention groups with T-test and with Significance Analysis of Microarrays (SAM). 19 genes were significantly different regulated by the two diets with a FDR of 23%. Most of thechanges in gene expression correlated with changes in blood lipids. Patients were examined in the morning after 12 h's fasting. Whole blood was obtained before (W0) and after completion (W8) of the dietary intervention for isolation of PBMCs and RNA extraction. From total RNA we prepared and hybridised biotinylated complementary RNA to GeneChip Human Gene 1.1 ST Arrays (Affymetrix Inc., Santa Clara, CA), and then washed, stained and scanned the slides using standardised protocols (Affymetrix Inc.). Signicance analysis of microarrays (SAM) was use to compare the difference in gene expression between groups.
Project description:Response of mouse mammary epithelial cells NMuMG to TGF-b1 - time course experiment. Identification of novel gene targets involved in TGF-b1-driven regulation of epithelial-mesenchymal transition (EMT).
Project description:TGF-b1-stimulation induces an epithelial dedifferentiation-process, throughout which epithelial cell sheets disintegrate and gradually switch into fibroblastic-appearing cells (EMT-like transition). Several transcription factors, some of them being TGF-b1-responsive, are functionally involved in such a switch and affect epithelial differentiation and plasticity. We used microarray-based gene expression profiling of mammary epithelial cells that actively undergo TGF-b1-induced epithelial dedifferentiation. Further, we determined gene expression changes in Basonuclin-1 knock-down cells in conjunction with TGF-b1-stimulation in order to determine a possible effect of Bnc1 on TGF-b1-responsive genes. Cells were transfected with non-silencing (control) siRNA or siRNA against Basonuclin-1 (Bnc1) for 24hrs. Subsequently, cells were treated with 5ng/ml TGF-b1 or left untreated for additional 24hrs. The experiments were performed as independent biological triplicate.
Project description:Recently, we found that a novel Traf2- and Nck-interacting kinase (TNIK) inhibitor, named NCB-0846, was capable of attenuating tumor-initiating cells among human colorectal cancer. The cross link between EMT and cancer stemness has been revealed in several studies and other group showed another TNIK inhibitor named KY-05009 had inhibited the TGF-β-induced EMT. Therefore we evaluated whether this small-molecule compound could have efficacy to inhibit TGF-β-induced EMT. NCB-0846 reduced the expression of mesenchymal markers (Vimentin and N-cadherin) and upregulated the expression of epithelial marker E-cadherin in A549 and H2228 non-small cell lung cancer cells. NCB-0846 suppressed the phosphorylation and nuclear translocation of Smad proteins and also inhibited migration, invasion, and metastasis. NCB-0846 inhibited TGF-β1-induced EMT through the down-regulation of TGF-β receptor-1 (TβRI) in mRNA levels. MiR-186-5p and miR-320 family were identified as candidate miRNAs that could target TβRI and we found that miR-186-5p and miR-320s inhibited TβRI expression. NCB-0846 might be a novel therapeutics drugs that targets the invasion and metastasis through inhibiting TGF-β-induced EMT in lung cancer. Overall design: EMT related markers and ability of migration, invasion, and metastasis were examined after exposure to TGF-B1 only or co-treated with TGF-B1 and NCB-0846 or NCB-970.
Project description:TGF-b1-stimulation induces an epithelial dedifferentiation-process, throughout which epithelial cell sheets disintegrate and gradually switch into fibroblastic-appearing cells (EMT-like transition). The purpose of these profiles was to identify differentially expressed genes that are regulated transcriptionally. Standard microarry-based gene expression profiles measure steady-state RNA but do not provide insight into underlying regulatory principles. NIAC-NTR-based gene expression profiling (Kenzelmann et al., PNAS, 2007) essentially enables the dissection of transcriptionally versus non-transcriptionally regulated genes within respective analysed time-frames. Briefly, NIAC-NTR relies on incorporation of 4sU (thio-uridine) into nascent RNA, which can subsequently be specifically isolated by custom-made columns. Total- and enriched (4sU-labeled) are then further processed for microarray gene expression profiling by standard procedures. This dataset complements previously released data of NIAC-NTR-based gene expression profiling of cells treated with TGF-b1 and 4sU for 2hrs [GSE23833]. The present data and files represent the outcome of NIAC-NTR-based gene expression profiling of cells treated with TGF-b1 for 24hrs and incubation with 4sU for the last 2hrs (22hrs-24hrs of TGF-b1-stimulation). NIAC-NTR: Non Invasive Application and Capture of Newly Transcribed RNA NMuMG cells were seeded 24hrs prior to treatment. Cells were stimulated with 5ng/ml TGF-b1 for a total of 24hrs. After 22hrs of stimulation 200µM 4sU thio-uridine was added to the cultures and further incubated for another 2hrs. Total RNA was extracted using RNeasy Mini Kits (Qiagen). 4sU-labeled RNA was further extracted from total RNA using mercury-based custom-made columns. The experiments were performed as independent biological triplicate. Details about isolation of 4sU-labeled RNA can be found in Kenzelmann et al. PNAS, 2007.
Project description:The Tgf-b signaling pathway plays an important role in both embryonic development and epithelial to mesenchymal transition (EMT), but the influence of this pathway and its relationship with EMT in fibroblast reprogramming is not defined. Using Affymetrix mouse genome array and mouse embryonic fibroblast cells (MEFs), we analyzed the expression profiles of Tgf-b1 and one of its important mediators in EMT, Snail, regulated genes at Day 10 during the process of reprogramming. A total of 535 genes were differentially expressed (2-fold change) in cells activated by Tgf-b1 and 970 genes were differentially expressed in cells over-expressing Snail. Among them, 170 genes were shared in both categories. The differentially expressed genes involve in many biological processes, including Tgf-b signaling pathway, cell communication, ECM-receptor interaction, cell adhesion, focal adhersion, and Hedgehog signaling pathway Overall design: MEFs derived from a OG2 mouse is a typical kind of fibroblast cells used in most reprogramming studies. Cells were cultured in MEF medium in an incubator with 5% CO2 at 37oC before retrovirus infection and the medium was changed into ES medium after infection of the reprogramming cocktail (Sox2, Klf4, Oct4 and cMyc). At Day 10 after infection, cells were collected and total RNA were extracted from the cells and was hybridized on Affymetrix GeneChip Mouse Genome 430 2.0 Array.
Project description:Background: Epithelial-to-mesenchymal transition (EMT) is considered an important driving mechanism behind aggressive cancer phenotype. This was recently challenged by the finding that cells can metastasize without undergoing EMT. However, the same studies confirmed the important role of the EMT program in drug resistance. The EMT program is largely dependent on the cell’s microenvironment. Acriflavine (ACF) is a heteroaromatic dye with antibacterial and antiviral effects. Recently, ACF was suggested as anticancer agent for its topoisomerase inhibitor activity. ACF further blocks the hypoxia-inducible factor (HIF) pathway, an important driver of cancer aggressiveness. How ACF works in cancer is however unknown. Aim: Identification of the working mechanism, molecular pathways and signaling of ACF in EMT cancer cells. To this end, three in vitro models were developed of EMT induction (human pancreatic cancer cells stimulated with TGF-b1, human pancreatic cancer cells stimulated with CoCl2, drug resistance against sorafenib in human liver cancer cells). Only the first model - PANC1 stimulated with TGF-b1 - is discussed in this GEO submission. Overall design: Five conditions were investigated: (1) untreated PANC1 cells (= controls), (2) PANC1 cells treated with TGFb1, (3) PANC1 cells treated with TGFb1 and ACF, (3) PANC1 cells treated with TGFb1 and Valproate, (5) PANC1 cells treated with ACF only. For each condition three biological replicates were profiled. For the control and the TGFb1 condition, a fourth biological replicate was profiled as well.