Investigations into the molecular regulation of gene expression in airway smooth muscle cells
The phenotype of airway smooth muscle (ASM) differs in asthmatic patients compared to healthy individuals. Hyperplasia, hypertrophy, increased contraction and altered synthetic capabilities of asthmatic airway smooth muscle cells (ASMCs) have all been reported, however, the molecular mechanisms underlying a number of these changes remain unclear. This project utilizes molecular techniques to investigate the regulation of gene expression in cultured ASMCs isolated from both healthy and asthmatic individuals. microRNAs (miRNAs) are short, non-coding RNAs that exert a post-transcriptional regulation on gene expression by targeting specific mRNAs for down regulation through either mRNA degradation or translational repression. During this project microarray studies have been performed to determine differences in the miRNA expression profile of cultured ASMCs isolated from healthy or moderate asthmatic volunteers. These studies have identified a number miRNAs with differential expression in asthmatic ASMCs compared to healthy controls. Of particular note, it is demonstrated that the abundance of both mature miR-155 and its host transcript, BIC, are approximately three-fold lower in cultured asthmatic ASMCs compared to healthy controls. Various experimental approaches to identify endogenous targets for miR-155 in cultured ASMCs are described. The advent of next-generation sequencing technology has had a great influence on the field of chromatin and transcriptional regulation. In particular it has been shown that specific histone modifications demarcate functional and architectural regions within the genome. Investigations using chromatin-immunoprecipitation followed by next-generation sequencing (ChiP-seq) to generate a profile of histone methylation enrichments across the genome of cultured healthy ASMCs are described. It is envisaged that this data set will provide a resource for researchers interested in the transcriptional regulation of gene expression in ASM, in particular it will facilitate the identification of novel distal elements involved in the regulation of specific loci.
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