A RasGAP-microRNA connection in cardiac hypertrophy

描述（由申请人提供）：在本提案中，我们将确定Ras GTP酶激活蛋白（RasGAP）在心肌肥大过程中调节microRNA-1（miR-1）的作用。MiR-1是基因表达的转录后调节因子，其在由工作负荷、生长因子或RasGAP诱导肥大时被急性下调，这是肌细胞生长所必需的结果。我们的初步数据显示，RasGAP SH 3结合蛋白（G3 BP）以RasGAP和Akt依赖的方式结合miR-1。我们假设肥大刺激诱导Akt介导的G3 BP磷酸化及其随后的RasGAP-细丝蛋白复合物的招募。这使其与miR-1非常接近，在那里它结合并水解未成熟的miR-1。随后，miR- 1的下调导致其靶点的上调，所述靶点包括：RasGAP、Cdk 9、纤连蛋白、内皮素和胰岛素样生长因子等。这些基因在心脏肥大的发展中起着关键作用。因此，我们的目的是：1）研究RasGAP介导的心肌细胞肥大过程中miR-1的下调机制。为此目的，我们将利用培养的肌细胞与重组cDNA、腺病毒和启动子构建体结合，使用拉伸作为肥大刺激以：a.检测G3 BP在miR-1转录后调节中的作用，B.检查Akt在RasGAP-G3 BP调节的miR-1稳定性中的作用，c.检查细丝蛋白-C在RasGAP-G3 BP募集中的作用，d.检查肥大和RasGAP激活途径对miR-1的转录与转录后调节的影响。目的2）研究RasGAP和miR-1在小鼠心肌肥厚中的作用。MicroRNA是新发现的心脏基因表达调节因子。它们为心血管疾病提供了潜在的有希望的治疗靶点，就像它们在癌症中所做的那样。但首先有必要确定它们的基因靶点，并了解它们的调节和功能机制。本研究拟探讨Ras GT3激活蛋白对miR-1的调控机制及其在心肌肥厚诱导中的作用。

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. PUBLIC HEALTH RELEVANCE MicroRNA are newly discovered regulators of gene expression in the heart. They provide potentially promising therapeutic targets in cardiovascular diseases as they do in cancer. But first it is necessary to identify their gene targets and understand the mechanism of their regulation and function. In this proposal we will investigate the mechanism of regulation of miR-1 by Ras GTPase activating protein and its role in induction of cardiac hypertrophy.