Identification of hypoxia regulated HIF-1A and HIF-2A specific target genes in Glioblastoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Koh Mei Yee
PROVIDER: S-ECPF-GEOD-27523 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells. 12 samples were analysed comprising 4 experimental conditions (normoxia scr, hypoxia scr, hypoxia siHIF1, hypoxia siHIF2) in triplicate. We made pairwise comparisons between the averages of each triplicate set to normoxia scr using the Partek suite.
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Project description:HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells.
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Project description:Cell lines. HEK293 human embryonic kidney cells were obtained from ATCC (American Type Culture Collection, Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Heat inactivated, Hyclone, Logan, Utah) and 1X antibiotics/glutamine (100 units/ml penicillin, 100ug/ml streptomycin, 292ug/ml L-Glutamine sulfate, all from Invitrogen Corp, Carlsbad, CA.), under either hypoxic (1% O2) or normoxic (21% O2) conditions at 37C in a tissue culture incubator. Treatment. Cells treated in hypoxia for 16 hours or transfected with HIF-1a (HIF-1amut or HIF2a) plasmid. At the end of treatment, cells were collected and washed. Microarray studies. Total RNA samples were extracted from HEK293T cells grown in 1% or 21% O2 overnight and from transfected cells. Total RNA (20 µg) from each sample was synthesized into double-stranded cDNA with reverse transcriptase (Fairplay labeling kit, Stratagen, La Jolla, CA) using an oligo d(T) primer. The double stranded cDNA from untreated cells was labeled with Cy3 monofunctional reactive dye and that from hypoxia-treated or transfected cells was labeled with Cy5 monofunctinal reactive dye. The probe was hybridized to a long oligo array (Hs-Operon V2-vB1.1px.gal) containing 20K human transcripts (NCI Microarray Facility, Advanced Technology Center, Gaithersburg, MD) overnight at 42C . The slides were washed and spin-dried. Microarray slides were scanned with a GenePix 4000 microarray scanner (Axon Instruments, Union City, CA.). The microarray images were analyzed with GenePix 3.0 software and data analysis was performed with the MicroArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at the NCI. Keywords: other
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Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells
2014-05-14 | E-GEOD-57613 | biostudies-arrayexpress
Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes.
2014-05-14 | GSE57613 | GEO
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