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。

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.