SMOOTH MUSCLE HYPERTROPHY REGULATED BY MICRORNAS AND THEIR TARGET GENES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 平滑肌（SM）肥大发生在各种内脏肌肉响应病理生理条件。我们建立了一种小肠部分梗阻（PO）模型，其中SM肥大是口服的，但不是对PO的位置，以研究肥大和/或增生过程中平滑肌细胞（SMCs）发生的重塑。microRNA（miRNAs）调节SMC中重要的细胞功能，如分化、增殖和凋亡。我们初步的微阵列分析显示，PO模型中的基因表达与对照肌肉的基因表达显著不同。我们还鉴定了一组独特的在小鼠和人类小肠中高度表达的miRNA，其中miR-143和miR-145特别丰富。这些miRNA也在分选的SMC中大量表达，并且两者在PO模型中下调。这导致了SM肥大部分受SMC依赖性miRNA调节的假设。为了确定miRNAs在小肠肥大发生中的作用，将解决以下三个目标：目标1：鉴定小肠SMC中表达的miRNAs以及随着SMC肥大的发生而发生的变化;目标2：鉴定小肠SMC中表达的信使RNA（mRNAs）以及随着肥大的发生而发生的变化;目标3：鉴定特异性调节肥大相关基因的miRNAs。一些分子，遗传学和生物信息学的方法将被用来开发一个功能之间的联系，在肥大的SMC的miRNA和靶基因的表达。对于目标1，将在我们的PO模型中研究SMC肥大，该模型在SM特异性Cre/eGFP小鼠中产生，其允许高度特异性SMC分选和454测序（焦磷酸测序）技术。对于目标2，我们将使用从分选的SMC分离的mRNA的基因芯片分析来鉴定肥大依赖性基因。对于目标3，我们将通过首先使用生物信息学将miRNA与靶基因相关联，然后使用尖端分子生物学技术验证这些关系来鉴定调节肥大基因的miRNA。我们将主要关注miR-143和miR-145及其靶基因，以了解它们如何在增生和/或肥大期间调节SMC的发育。了解SMCs中基因表达的变化为了解SM病理生理条件下表型的调节提供了令人兴奋的新机会。鉴定与肥大相关的遗传标记将有助于开发miRNA药物，这些药物有可能使导致肥大的mRNA靶点正常化，从而逆转这些疾病中发生的一些不必要的病理变化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.