Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human breast cancer cell line MDA-MB-231 treated with T025 against untreated controls

ABSTRACT: Deep RNA sequencing was performed to investigate the impact of CLKs inhibitor, T-025, on RNA splicing in triple-negative breast cancer cell lines. A total of 30 million MDA-MB-231 cells were seeded in a 6-well plate. The following day, cells were treated with 1 µM T-025 or 0.1% DMSO as negative control for 24 hours. Samples from 3 biological replicates were used. Total RNA was isolated using the RNeasy plus mini kit (Qiagen). Stranded PolyA-selected libraries were prepared using the DNBseq platform, and 100 M 150-bp paired-end reads were sequenced on a DNBSEQ-G400 sequencer by BGI Europe. The differentially expressed genes (DEGs) and alternative splicing events (ASEs) were analyzed to unravel the transcriptomic changes in TNBC cells upon T-025 treatment.

INSTRUMENT(S): DNBSEQ-G400

ORGANISM(S): Homo sapiens

SUBMITTER: Jurjun Velde

PROVIDER: E-MTAB-14664 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

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RNA-seq of human breast cancer cell line MDA-MB-231 treated with T025 against untreated controls

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