Gene expression profile of HCT116 exposed to chronic hypoxia
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ABSTRACT: To identify genes involved in survival to prolonged hypoxia we exposed HCT116 to hypoxia for 3 days. Control cells were exposed to normoxic conditions. HCT116 colon cancer cells were serum starved and exposed to hypoxia (1%O2) or normoxia (21%O2) for 3 days.
Project description:To determine the effects of depleting TIP60, CDK8, or HIF1A on the transcriptional response to hypoxia, we performed RNAseq analysis of four HCT116 colorectal carcinoma cell lines (shNT, HIF1A-/-, shTIP60 and shCDK8) in normoxic and hypoxic (24hrs, 1% O2) conditions. PolyA RNA for two independent biological replicates was purified from HCT116 cells stably expressing an shRNA against a non-targeting control (shNT), TIP60 (shTIP60) or CDK8 (shCDK8), or genetically deleted HIF1A (HIF1A-/-) subjected to 24hrs 1% O2 (hypoxia) or maintained under ambient oxygen (21%; normoxia) was sequenced on the Ion Torrent platform. Reads were aligned to the human genome and gene-level counts were used for differential expression analysis.
Project description:To test if CDK8 acts directly at HIF1A target genes, we performed ChIP-seq experiments in HCT116 cells under normoxic and hypoxic conditions. ChIP-seq for CDK8 versus Input under normoxia and 24hrs hypoxia (1% O2).
Project description:To define the contribution of CDK8 versus CDK19 to gene expression control, we performed a series of microarray assays for cells where each kinase was stably knocked down. Toward this end, we subjected HCT116 cells to three different stress stimuli: 5-fluorouracil (5FU), glucose deprivation, and hypoxia. We found that CDK8, but not CDK19, functions as a widespread coactivator of HIF1A target genes in hypoxia. Total RNA from HCT116 cells harvested using an RNeasy kit (Qiagen) was used for gene expression analysis on Affymetrix HuGene 1.0 ST arrays following the manufacturer’s instructions. Differential gene expression was determined with Partek software using one-way ANOVA.
Project description:To identify genes involved in survival to prolonged hypoxia we exposed HCT116 to hypoxia for 3 days. Control cells were exposed to normoxic conditions.
Project description:To determine the effects of depleting TIP60, CDK8, or HIF1A on the transcriptional response to hypoxia, we performed RNAseq analysis of four HCT116 colorectal carcinoma cell lines (shNT, HIF1A-/-, shTIP60 and shCDK8) in normoxic and hypoxic (24hrs, 1% O2) conditions.
Project description:Hypoxia is associated with poor prognosis in most solid tumors due to its multiple effects on therapy resistance, angiogenesis, apoptotic resistance, and tumor invasion/metastasis. Here we used a comprehensive omics profiling to investigate hypoxia-regulated gene expression in HCT116 colon cancer cells. Quantitative analyses of proteome and secretome were performed in HCT116 cells cultured under hypoxic or normoxic conditions. A total of 5,700 proteins were quantified in proteome analysis and 722 proteins were quantified in secretome analysis. Both datasets were combined with the transcriptome and translatome datasets for further analysis. Verification of candidate proteins/genes in this integrated omics analysis was performed using immunoblotting and quantitative real-time RT-PCR analyses. We also performed polysome profiling to assess changes in translational efficiency of hypoxia-induced genes. Notably, several genes were differently regulated at the transcriptional and translational levels in HCT116 cells during hypoxia. Bioinformatics analysis suggested that hypoxia regulates translation of genes involved in extracellular matrix organization, extracellular exosomes, and protein processing in endoplasmic reticulum. Aberrations in these metabolic pathways appear to be correlated with an increased risk of tumor invasion/metastasis.
Project description:Systemic arterial smooth muscle cells are exposed to a broad range of oxygen concentrations under physiological conditions. Hypoxia can modulate the proliferative response of smooth muscle cells leading to speculation about its role in vasculogenesis, vascular remodelling and the pathogenesis of arterial disease. The effect of hypoxia has been inconsistent, however, with both enhanced proliferation and growth arrest reported. Nevertheless, these reports support an important effect of hypoxia on smooth muscle cell proliferation and, given its physiological and clinical relevance, this requires clarification. We posited that variation in O2 concentration, within the range that exists in vivo, may have different effects on the proliferation and survival of vascular smooth muscle cells. Experiment Overall Design: Human aortic smooth muscle cells (HASMC) were propagated to passage 6 in SMGM-2 medium reached 80% confluence, the media was changed and the cells were incubated for a further 16 hrs or 48 hrs under either normoxic or hypoxic conditions (1% and 3%O2 ).
Project description:Genome wide analysis RNA polymerase II (RNA Pol II) and histone marker H3K4me3 in hypoxia and normoxia using ChIP-seq. Breast cancer cell line (MCF-7) is cultured in normoxic condition (21% O2) and hypoxic condition (0.5%O2) for 16 hours. Immunoprecipitation of RNA Pol II and H3K4me3.
Project description:To delineate the role of hypoxia in esophageal epithelial biology, we carried out gene array experiments using a non-transformed immortalized diploid human esophageal cell line, EPC2-hTERT (Mol Cancer Res. 2003;1:729-38). Unlike cancer cell lines, EPC2-hTERT has no genetic alterations at early passages that may affect the cellular response to hypoxia. Experiment Overall Design: EPC2-hTERT cells were exposed to moderate (1% O2) hypoxia in experiment 1 (Exp1) or severe (0.2% O2) hypoxia in experiment 2 (Exp2). Normoxia (21% O2) served as a control in both experiments.
Project description:To define ncRNA expression in hypoxic endothelial cells, we applied pro-angiogenic hypoxia to cultured endothelial cells. Afterwards, total RNA was isolated and underwent RNA-seq analysis. HUVECs were subjected to normoxic or hypoxic (0.1-0.2% O2) cell culture conditions.