Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of IEC4.1 cells in response to TNFα stimulation.

ABSTRACT: Mouse intestinal epithelial cells (IEC4.1 cells) were stimulated with TNFα (10 ng/ml) for 4h. The Agilent SurePrint G3 Mouse Gene Expression Microarray (G4852A) was used for the analysis, which provides full coverage of genes and transcripts with the most up-to-date content, including mRNAs and lincRNAs (http://www.chem.agilent.com/store/en_US/Prod-G4852A/G4852A). IEC4.1 cells were grown to 80% confluence for four groups: the siRNA control (Group A, cells treated with a non-specific scrambied siRNA control), the TNFα -stimulated (Group B, cells treated with the siRNA control plus TNFα stimulation), lincRNA-Cox2 siRNA (Group C, cells treated with an siRNA to lincRNA-Cox2), and lincRNA-Cox2 siRNA/ TNFα stimulated (Group D, cells treated with the lincNRA-Cox2 siRNA plus TNFα stimulation). Cells were treated with the siRNAs for 24h, followed by additional culture for 4h in the presence or absence of TNFα (10 ng/ml). Total RNAs were prepared with the RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction (Ambion).

ORGANISM(S): Mus musculus

SUBMITTER: Xianming Chen

PROVIDER: E-MTAB-3904 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:While thousands of long non-coding RNAs (lncRNAs) are expressed in higher eukaryotes, the potential regulatory roles of lncRNAs in regulated gene transcription programs remain rather poorly understood. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the androgen receptor (AR) and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM, to the N-terminally methylated AR. Unexpectedly, recognition of the H3K4me3 promoter mark by the PHD finger-domain of Pygopus2, recruited by PCGEM1, proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells, revealing a novel aspect of ligand-induced enhancer-promoter interactions. In M-bM-^@M-^\resistantM-bM-^@M-^] prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for the robust activation of both truncated and full length AR, causing DHT-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA)-mediated targeting of these lncRNAs in these resistant cancer cell lines strongly suppressed xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors. Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq); ChIRP-seq data for a lincRNA (PCGEM1). LNCaP cells were grown to 30-50% confluence and siRNA/ASO transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturerM-bM-^@M-^Ys instructions. Control samples were transfected with scramble ASO and control siRNA, respectively. On the following day of transfection, the cells were cultured in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM for 1 hour we have sequenced the DNA sequences that are associated with the presence of a RNA molecule on a genome wide scale. PCGEM1 probe, -DHT PCGEM1 probe, +DHT

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Transcriptome analysis of IEC4.1 cells in response to TNFα stimulation.

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