RNAi profiling by array of human Ewings carcinoma cells to down regulate EWS-FLI1
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Gong-Hong Wei
PROVIDER: E-MEXP-2759 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Goal: identification of differentially expressed genes after STEAP1 silencing. Ewing tumors (ET) are characterized by oncogenic EWS/ETS translocations and early metastasis. STEAP1 is a membrane-bound channel protein of unkown function. While overexpressed in many cancers, STEAP1 expression is strongly restricted to mesenchymal stem cells, prostate and urothelium among benign tissues. Here we show that STEAP1 is a direct transcriptional target of EWS/FLI1 and critical for ET malignancy. We demonstrate that STEAP1 is most prominently expressed in ET among sarcomas and provide further evidence for the concept of STEAP1 as a universal diagnostic marker for carcinomas. Using RNA interference we determined that STEAP1 promotes cellular invasiveness and anchorage-independent growth in vitro and accelerates tumor growth and metastasis in vivo. Transcriptome and proteome analyses as well as functional studies reveal that STEAP1 contributes to the generation of reactive oxygen species that in turn regulate the levels of redox-sensitive signaling molecules and pro-metatstatic genes. In synopsis, these data illuminate the hitherto unkown oncogenic function of STEAP1 as a redox-modulator in ET and point to a potential role of STEAP1 as universal drug target for anti-cancer therapy. 6 samples (3x A673 cells; 3x SK-N-MC cells); for each cell line one sample was transfected with control non silencing siRNA and two samples with different STEAP1 siRNAs (siSTEAP1_2 and siSTEAP1_3).
2012-01-01 | E-GEOD-26422 | biostudies-arrayexpress
Project description:Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome-depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately transcriptional dysregulation. Examination of two transcription factors in two different cell types, as well as three histone methylation marks and FAIRE
2011-11-09 | E-GEOD-31838 | biostudies-arrayexpress
Project description:EWS-FLI1 is a chimeric ETS transcription factor that is, due to a chromosomal rearrangement, specifically expressed in Ewingâs sarcoma family tumors (ESFT) and is thought to be the initiating event in the development of the disease. Previous genomic profiling experiments have identified a number of EWS-FLI1 regulated genes and genes that discriminate ESFT from other sarcomas, but so far a comprehensive analysis of EWS-FLI1 dependent molecular functions characterizing this aggressive cancer is lacking. In this study a molecular function map of ESFT was constructed based on an integrative analysis of gene expression profiling experiments on a uniform microarray platform following EWS-FLI1 knockdown in a panel of five ESFT cell lines, and on gene expression data from the same platform of 59 primary ESFT tumors. Based on the assumption that EWS-FLI1 is the driving transcriptional force in ESFT pathogenesis, we predicted an inverse correlation of gene expression for EWS-FLI1 regulated genes between the putative tissue of origin and the cell lines under EWS-FLI1 knockdown conditions. Consistent with recent reports, mesenchymal progenitor cells (MPC) were found to fit this hypothesis best and were therefore used as the reference tissue for the construction of the molecular function map in ESFT. The interrelations of molecular pathways were visualized by measuring the similarity among annotated gene functions by gene sharing. The molecular function map highlighted distinct clusters of activities for EWS-FLI1 regulated genes in ESFT and revealed a striking difference between EWS-FLI1 up- and down-regulated genes: EWS-FLI1 induced genes mainly belong to cell cycle regulation, proliferation and response to DNA damage, while repressed genes were associated with differentiation and cell communication. This study revealed that EWS-FLI1 combines by distinct molecular mechanisms two important functions of cellular transformation in one protein, growth promotion and differentiation blockage. By taking MPC as a reference tissue a significant EWS-FLI1 signature was discovered in ESFT that only partially overlapped with previously published EWS-FLI1 dependent gene expression patterns, identifying a series of novel targets for the chimeric protein in ESFT. Our results may guide target selection for future ESFT specific therapies. Experiment Overall Design: EWS-FLI1 knock down was performed by expressing specific siRNAs against EWS-FLI1 as small hairpin (sh) RNAs from pSUPER-based retroviral expression constructs in 5 different Ewing's sarcoma cell lines (WE68, SK-N-MC, TC252, STA-ET-1, STA-ET-7.2). As a negative control in each cell line, pSTNeg (Ambion, Applied Biosystems, Brunn am Gebirge, Austria) encoding a scrambled shRNA with no significant similarity to human sequences was used.
2009-05-11 | E-GEOD-14543 | biostudies-arrayexpress
Project description:ETS gene fusions have been characterized in a majority of prostate cancers, however key molecular alterations in ETS negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers. Keywords: Genetic Modification 22RV1 cells were infected with non-targeting siRNA or siRNA against SPINK1. For reported hybridizations, the reference channel is 22RV1 cells infected with non-targeting siRNA. Duplicate hybridizations were performed with duplicate dye flips, for a total of four arrays. Over and under-expressed signatures were generated by filtering to include only features with significant differential expression (PValueLogRatio < 0.01) in all hybridizations and Cy5/Cy3 ratios > or < 1 in all hybridizations.
2008-06-10 | E-GEOD-11132 | biostudies-arrayexpress
Project description:We have examined the biological effect of EWS/ETS in human MPCs using UET-13 cells that are obtained by prolonging the lifespan of human bone marrow stromal cells using the retroviral transgenes hTERT and E7. By exploiting tetracycline-inducible systems for expressing EWS/ETS (EWS/FLI1 and EWS/ERG), we investigated candidates for genes whose expression is regulated by EWS/ETS in human MPCs. Experiment Overall Design: A change of expression profiles in the presence of EWS/ETS using Affymetrix HG-U133 Plus 2.0 (GPL570).
2008-06-16 | E-GEOD-8665 | biostudies-arrayexpress
Project description:The outcome of infections with Toxoplasma gondii in humans is dependent in part on the genetic makeup of the infecting organism. Recent studies have indicated that most infecting Toxoplasma organisms fall into 1 of 3 canonical lineages. Previous studies have investigated the effects of Toxoplasma on its host cell transcriptome. Little is known, however, about the effects of three canonical lineages on brain cells, the principal site of parasite lifelong persistence. In this study, we examined the transcriptional profile of human neuroepithelioma cells in response to T. gondii infection using microarray analysis to characterize the strain-specific host cell response to 3 canonical T. gondii strains. We found that the extent of the expression changes varied considerably among the three strains. Neuroepithelial cells infected with type I exhibited the most differential gene expression, whereas type II infected cells had a substantially smaller number of genes which were differentially expressed. Cells infected with type III exhibited intermediate effects on gene expression. The three strains also differed in the individual genes and gene pathways which were altered following cellular infection. For example, gene ontology (GO) analysis indicated that type I infection largely affects genes related to central nervous system while type III infection largely alters genes which affect nucleotide metabolism; type II infection does not alter expression of a clearly defined set of genes. Moreover, Ingenuity pathway analysis (IPA) revealed the sophistication of different strain in its interactions with the host. These differences may explain some of the variation in the neurobiological effects of different strains of Toxoplasma on infected individuals. We infected SK-N-MC cells with 3 canonical Toxoplasma strains and assessed their gene expression in RNA at 20 hours post-infection using Affymetrix GeneChip. Infections and controls (no tachyzoites) were performed in duplicate and experiments for each strain were carried out on three separate occasions in order to include both technical replicates and biological replicates.
2011-06-24 | E-GEOD-22986 | biostudies-arrayexpress
Project description:The pesticide rotenone, a neurotoxin that inhibits the mitochondrial complex I, and destabilizes microtubules (MT) has been linked to Parkinson disease (PD) etiology and is often used to model this neurodegenerative disease (ND). Many of the mechanisms of action of rotenone are posited mechanisms of neurodegeneration; however, they are not fully understood. Therefore, the study of rotenone-affected functional pathways is pertinent to the understanding of NDs pathogenesis. This report describes the transcriptome analysis of a neuroblastoma (NB) cell line chronically exposed to marginally toxic and moderately toxic doses of rotenone. The results revealed a complex pleiotropic response to rotenone that impacts a variety of cellular events, including cell cycle, DNA damage response, proliferation, differentiation, senescence and cell death, which could lead to survival or neurodegeneration depending on the dose and time of exposure and cell phenotype. The response encompasses an array of physiological pathways, modulated by transcriptional and epigenetic regulatory networks, likely activated by homeostatic alterations. Pathways that incorporate the contribution of MT destabilization to rotenone toxicity are suggested to explain complex I-independent rotenone-induced alterations of metabolism and redox homeostasis. The postulated mechanisms involve the blockage of mitochondrial voltage-dependent anions channels (VDACs) by tubulin, which coupled with other rotenone-induced organelle dysfunctions may underlie many presumed neurodegeneration mechanisms associated with pathophysiological aspects of various NDs including PD, AD and their variant forms. Thus, further investigation of such pathways may help identify novel therapeutic paths for these NDs. SK-N-MC cells were grown in three different conditions: media + vehicle (EtOH), media + a sublethal dose (5 nM) of rotenone, and media + a slightly toxic dose (50 nM) of rotenone. Gene expression was examined at 1 and 4 weeks of rotenone treatment. Three experiments were performed, each lasting 1 and 4 weeks. For each experiment, 5 separate dishes of vehicle-treated, and rotenone-treated cells were harvested at 1 and 4 weeks (30 independent samples at each time point) and pooled into 3 samples (vehicle treated,and rotenone treated with 5 nM and 50 nM) at each time point for a total of 18 individual samples from all thre experiments. Total RNA isolated from each of the 18 individual samples was used to prepared labeled cRNA and hybridized to one Genechip (Affymetrix) Human Genome array HG-U133A at the UCLA microarray core facility (http://microarray.genetics.ucla.edu/). After QC check, the data was normalized and used to assess expression indexes and fold changes (FC > 2.0, compared to vehicle-treated controls) using the model-based expression indexes (MBEI) method implemented in dCHIP, ( http://biosun1.harvard.edu/complab/dchip/), and the Signifiance Analysis of Microarrays (SAM) software for multiple test corrections. Enrichment analysis was performed by the functional annotation tools implemented in DAVID ( http://david.abcc.ncifcrf.gov), to ascertain sets of rotenone DRGs that are enriched in certain biological annotations.
2012-08-08 | E-GEOD-35642 | biostudies-arrayexpress
Project description:HeLa cell line was trasfected by a EWS siRNA oligo causing knock-down of EWS or a siRNA scrambled oligo Three biological replicates were labeled in direct and dye-swap microarray experiments and hybridized onto an Agilent custom splicing-sensitive microarray platform.
2011-09-19 | E-GEOD-22929 | biostudies-arrayexpress
Project description:We show that EWS-FLI1, an aberrant transcription factor responsible for the pathogenesis of Ewing sarcoma, reprograms gene regulatory circuits by directly inducing or directly repressing enhancers. At GGAA repeats, which lack regulatory potential in other cell types and are not evolutionarily conserved, EWS- FLI1 multimers potently induce chromatin opening, recruit p300 and WDR5, and create de novo enhancers. GGAA repeat enhancers can loop to physically interact with target promoters, as demonstrated by chromosome conformation capture assays. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors and abrogating p300 recruitment. ChIP-seq for of 4 histone modifications (H3K27ac, H3K4me1, H3K4me3 and H3K27me3), FLI1, p300, WDR5, ELF1 and GABPA in primary Ewing sarcomas, Ewing sarcoma cell lines (A673 and SKMNC cells), and mesenchymal stem cells (MSC). EWS-FLI1 was knocked down in Ewing sarcoma cell lines with lentiviral shRNAs (shFLI1 and shGFP control). EWS-FLI1 was expressed in MSCs with lentiviral expression vectors (pLIV EWSFLI1 or pLIV empty vector control). * Raw data not provided for the MSC and Primary Ewing sarcoma samples. *
2014-10-30 | E-GEOD-61944 | biostudies-arrayexpress
Project description:Background: The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear. Results: We report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors. Conclusions: Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases. This is one part of the study that involved the examination of HepG2 gene expression. Biological triplicate Illumina Sentrex bead arrays.
2010-09-30 | E-GEOD-24419 | biostudies-arrayexpress