Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from human carcinoma (MCF7) derived cells that have been exposed to insulin analogues

ABSTRACT: Insulin analogues are designed to improve the pharmacokinetic parameters compared to regular human insulin. This provides a sustained control of blood glucose levels in diabetic patients. All novel insulin analogues are tested for their mitogenic side effects, however these assays do not take into account the molecular mode-of-action of different insulin analogues. Insulin analogues can bind the insulin receptor (INSR) and the insulin-like growth factor-1 receptor (IGF1R) with different affinities and consequently will activate different downstream signaling pathways. Here we used a panel of MCF7 human breast cancer cell lines that selectively express either one of the isoforms of the INSR (IRA or IRB) or the IGF1R. We sought to study the role of the different receptors (IRA, IRB and IGF1R) in the mitogenic signaling of insulin-like molecules (including insulin, glargine, X10 (or AspB10) and IGF1). MCF7 IRA, MCF7 IRB or MCF7 IGF1R cells (as described in Arch Toxicol. 2014 Apr;88(4):953-66. doi: 10.1007/s00204-014-1201-2. Epub 2014 Jan 25.) were cultured in RPMI supplemented with 5% (v/v) CDFBS (Hyclone) and used for experiments. Cells have been exposed for 1 or 6 hours to 10 nM of the indicated insulin-like molecule. As a control sample a vehicle stimulation was performed that contained everything except the active compound.

ORGANISM(S): Homo sapiens

SUBMITTER: Steven Wink

PROVIDER: E-GEOD-65398 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Despite the prevalence and biological relevance of both signalling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signalling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis of changes in alternative splicing using splicing-sensitive microarrays, induced by activation of two distinct signalling pathways, insulin and wingless, in Drosophila cells in culture. Results: Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and posttranscriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related with carbohydrate metabolism and cellular signalling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5â splice sites. Conclusion: Taken together, our data indicate that signalling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for cross-talk between different signalling pathways. To monitor transcriptional and alternative splicing changes induced by activation of the insulin and wingless pathways, a custom-designed microarray platform was employed featuring probes for all Drosophila genes for which different mRNA isoforms generated by alternative splicing have been described (see Blanchette M, Green RE, Brenner SE, Rio DC: Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 2005, 19(11):1306-1314.). Three biological replicates of total RNA isolated after pathway activation or controls (untreated cells for insulin, control dsRNA for wingless) were purified, reverse transcribed into cDNA and labelled with Cy5 or Cy3 fluorochromes and the cDNA was hybridized to the microarray,