SMOOTH MUSCLE HYPERTROPHY REGULATED BY MICRORNAS AND THEIR TARGET GENES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Smooth muscle (SM) hypertrophy occurs in a variety of visceral muscles in response to pathophysiological conditions. We have developed a small intestine partial obstruction (PO) model in which the SM hypertrophy is oral but not aboral to the site of PO, to study the remodeling that occurs in smooth muscle cells (SMCs) during hypertrophy and/or hyperplasia. microRNAs (miRNAs) regulate vital cellular functions in SMCs such as differentiation, proliferation, and apoptosis. Our preliminary microarray analysis reveals that gene expression in the PO model is dramatically different from that of control muscles. We have also identified a unique set of miRNAs that are highly expressed in the small intestine of mice and humans with miR-143 and miR-145 being particularly abundant. These miRNAs are also abundantly expressed in sorted SMCs and both are down-regulated in the PO model. This has led to the hypothesis that SM hypertrophy is regulated, in part, by SMC-dependent miRNAs. To determine the role of miRNAs in the development of small intestine hypertrophy, the following three aims will be addressed: Aim 1: Identify the miRNAs expressed in small intestine SMCs and the changes which occur with the development of SMC hypertrophy; Aim 2: Identify the messenger RNAs (mRNAs) expressed in small intestine SMCs and the changes which occur with the development of hypertrophy; Aim 3: Identify the miRNAs specifically regulating the genes associated with hypertrophy. Several molecular, genetic, and bioinformatic approaches will be used to develop a functional link between miRNA and target gene expression in the hypertrophic SMCs. For Aim 1, SMC hypertrophy will be studied in our PO model generated in the SM-specific Cre/eGFP mice which permit highly specific SMC sorting and 454 sequencing (pyrosequencing) techniques. For Aim 2, we will identify hypertrophy-dependent genes using GeneChip analyses of the mRNAs isolated from sorted SMCs. For Aim 3, we will identify the miRNAs regulating hypertrophy genes by first relating miRNAs to target genes using bioinformatics and then validating these relationships using cutting edge molecular biology techniques. We will primarily focus on miR-143 and miR-145 and their target genes to see how they regulate the development of SMCs during hyperplasia and/or hypertrophy. Understanding changes in gene expression in SMCs provides an exciting new opportunity for understanding the regulation of phenotype in SM pathophysiological conditions. Identifying the genetic markers associated with hypertrophy will aid in the development of miRNA drugs that have the potential to normalize mRNA targets that are responsible for hypertrophy and thus reverse some of the unwanted pathological changes that occur in these disorders.