A RasGAP-microRNA connection in cardiac hypertrophy

描述(申请人提供)：在这项提案中，我们将确定Ras GTP酶激活蛋白(RasGAP)在心肌肥厚过程中调节microRNA-1(miR-1)的作用。MIR-1是一种转录后基因表达调节因子，在工作超负荷、生长因子或RasGAP诱导肥大时急剧下调，这是心肌细胞生长所必需的结果。我们的初步数据表明，RasGAP SH3结合蛋白(G3BP)以RasGAP和Akt依赖的方式与miR-1结合。我们假设，肥大刺激诱导Akt介导的G3BP磷酸化，并随后被RasGAP-丝氨酸复合体募集。这使得它非常接近miR-1，在那里它结合并水解早熟的miR-1。随后，miR-1的下调导致其靶标上调，其中包括：RasGAP、CDK9、纤维连接蛋白、内皮素和胰岛素样生长因子等。这些基因在心肌肥厚的发生发展中起着关键作用。因此，我们的目标是：1)研究RasGAP介导的miR-1在心肌细胞肥大过程中下调的机制。为此，我们将利用培养的心肌细胞与重组cDNA、腺病毒和启动子构建物结合，使用STRAND作为肥大刺激：A.检测G3BP在miR-1转录后调控中的作用，B.检测Akt在RasGAP-G3BP调控的miR-1稳定性中的作用，C.研究细丝-C在RasGAP-G3BP招募中的作用，D.研究肥大和RasGAP激活的途径对miR-1转录和转录后调控的影响。目的2)研究RasGAP和miR-1在小鼠心肌肥厚中的作用。

DESCRIPTION (provided by applicant): In this proposal we will determine the role of Ras GTPase-activating protein (RasGAP) in regulating microRNA-1 (miR-1) during cardiac hypertrophy. MiR-1 is a post- transcriptional regulator of gene expression that is acutely down-regulated upon induction of hypertrophy by work overload, growth factors, or RasGAP, an outcome that is necessary for myocyte growth. Our preliminary data show that RasGAP SH3-binding protein (G3BP) binds miR-1 in a RasGAP- and Akt-dependent manner. We hypothesize that hypertrophic stimuli induce Akt-mediated G3BP phosphorylation and its subsequent recruitment by RasGAP-filamin complex. This brings it into close proximity to miR-1, where it binds and hydrolyzes premature miR-1. Subsequently, down-regulation of miR- 1 results in upregulation of its targets that include: RasGAP, Cdk9, fibronectin, endothelin, and insulin-like growth factor, among others. These genes play a critical role in the development of cardiac hypertrophy. Thus, our Aims are: 1) to study the mechanism of RasGAP-mediated down-regulation of miR-1 during myocyte hypertrophy. For this aim we will utilize cultured myocytes in conjunction with recombinant cDNA, adenoviruses, and promoter constructs, using stretch as a hypertrophic stimulus to: a. examine the role of G3BP in post-transcriptional regulation of miR-1, b. examine the role of Akt in RasGAP-G3BP-regulated miR-1 stability, c. examine the role of filamin-C in recruitment of RasGAP-G3BP, d. examine the effect of hypertrophy and the RasGAP- activated pathway on transcriptional vs. post-transcriptional regulation of miR-1. Aim 2) to study the role of RasGAP and miR-1 during cardiac hypertrophy in a mouse model.