Unknown,Transcriptomics,Genomics,Proteomics

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Identification of target genes regulated by M-NM-^TNp63M-NM-1 in SaOS-2 cells stably expressed with M-NM-^TNp63M-NM-1

ABSTRACT: To identify the target gene of M-NM-^TNp63M-NM-1, in osteosarcoma cells, we performed the global gene expression profiling in SaOS- 2 cells stably overexpressing the M-NM-^TNp63M-NM-1 gene (SaOS- 2-M-NM-^TNp63M-NM-1). M-NM-^TNp63M-NM-1 target genes were identified by comparison with genes expressed from SaOS-2-EV transfected with an empty vector ( SaOS-2-EV). Total RNA was extracted from SaOS- 2-M-NM-^TNp63M-NM-1 and SaOS-2-EV cells cultured in three independent replicates for each condition. Cy3-labeled cRNA was synthesized and hybridized to Agilent human 8 x 60K microarrays. Raw data, obtained after array image analysis were normalized and used to retrieve differentially expressed genes in M-NM-^TNp63M-NM-1 overexpressing SaOS- 2 cells and genes from control SaOS-2-EV cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Ram Mohan Ram Kumar

PROVIDER: E-GEOD-54942 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mammalian FoxO transcription factors - FoxO1, FoxO3, FoxO4 - function in the nucleus to direct transcription of specific gene targets governing cellular survival, proliferation, metabolism, differentiation and oxidative defense. Activation of PI3K by extracellular growth factors leads to AKT-mediated phosphorylation of FoxO1, FoxO3 and FoxO4, resulting in their sequestration in the cytoplasm such that they are unable to regulate their gene targets. Our study identified FoxOs as novel tumor suppressors in kidney cancer (Gan et al, 2010, Cancer Cell). To understand the tumor suppression function of FoxOs in kidney cancer cells, we performed gene expression profiling in human kidney cancer cells upon FoxO1 or FoxO3 reactivation in order to identify the key transcriptomic alterations mediating FoxO tumor suppression function in kidney cancer cells. We generated RCC4 and UMRC2 cell lines (two human kidney cancer cells with low endogenous FoxO1 and FoxO3 expression) with stable expression of FoxO1(TA)ERT2 or FoxO3(TA)ERT2 construct, which expressed a fusion protein consisting of FoxO(TA) (containing three Ser/Thr AKT phosphorylation sites mutated to alanine) fused to the T2-modified estrogen receptor (ERT2) moiety. We documented that the FoxO(TA)ERT2 fusion protein sequestered FoxO(TA) in the cytoplasm and that 4OHT treatment resulted in rapid translocation of FoxO(TA)ERT2 into the nucleus. We also established stable cell lines with ERT2 expression as control cell lines. (For simplicity, ERT2, FoxO1(TA)ERT2 and FoxO3(TA)ERT2 cell lines will be referred to as EV (empty vector), FoxO1 and FoxO3, respectively, hereafter). We then conducted comparative transcriptome analysis (using the Human Genome U133 Plus 2.0 Array) of EV, FoxO1, or FoxO3-expressing RCC4 and UMRC2 cells at 12 hours with or without 100 nm 4OHT treatment (cultured in DMEM+10% FBS with puromycin selection). To enrich for more proximal actions of FoxO, we selected the 12 hour time point as time course studies revealed dramatic transcriptional changes of known FoxO targets (such as Cyclin D1), yet no discernable cellular phenotypes (apoptosis and cell cycle arrest). We generated 4 transcriptome datasets: FoxO1 RCC4, FoxO3 RCC4, FoxO1 UMRC2, and FoxO3 UMRC2 (by comparing transcriptome data with or without 4OHT treatment), and normalized these transcriptome data against 4OHT-treated EV cells, which show modest 4OHT-induced transcriptional changes.

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Identification of target genes regulated by M-NM-^TNp63M-NM-1 in SaOS-2 cells stably expressed with M-NM-^TNp63M-NM-1

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