The role of microRNA210 in cardiomyocyte response to ischemia-reperfusion

描述（由申请人提供）：缺血性心脏病（IHD）是发达国家的主要流行病，然而，导致IHD中心肌细胞死亡的分子机制尚未完全了解。microRNA（miRNAs）是一类短的非编码RNA，通过与靶mRNA结合并引起降解或翻译抑制来调节基因表达。我们最近发现，暴露于缺氧的心肌细胞中miRNA-210（miR-210）的水平升高。miR-210对缺氧的反应在p53-/-小鼠胚胎成纤维细胞（MEF）中减弱，暗示p53在miR-210的调节中的作用。然而，目前还不清楚miR-210在心肌细胞对缺氧的反应中起什么作用。此外，尽管miR-210和缺氧诱导因子（HIF）都被缺氧激活，并且HIF调节miR-210表达，但miR-210对HIF激活的生物学效应的相对贡献尚不清楚。在本研究中，我们将阐明缺氧对miR-210的调节机制，并确定miR-210在心肌细胞对缺血再灌注（I/R）反应中的作用。我们的中心假设是miR-210在转录水平上受p53调控，减少心肌细胞对I/R的死亡反应，并介导HIF的一些生理作用。在目标1中，我们将确定miR-210是否在I/R引起的心肌细胞死亡和ROS产生中发挥作用。除了体外研究，我们还将使用金缀合纳米颗粒的新方法研究miR-210 KO小鼠和心脏miR-210敲低小鼠。我们将这些小鼠进行I/R，然后测量细胞死亡和缺血性损伤。在目标2中，我们将确定miR-210在HIF激活的生物学效应中的作用。我们将在HIF 11-/- MEFs中过表达miR-210，然后测量响应缺氧的各种生理参数。我们还将使用心脏特异性Von Hippel-Lindau（VHL）基因敲除小鼠，这些小鼠具有组成性活性HIF并显示出几种异常，包括心房肿瘤形成，脂质积聚和心肌病。我们将VHL-/-小鼠与miR-210敲除动物杂交，并将评估miR-210敲除是否会改变心脏中慢性HIF激活的变化。最后，在目标3中，我们将阐明p53调控miR-210的机制。我们将通过测量p53-/- MEFs中的miR-210前体分子并进行ChIP和核运行试验来评估p53是否在转录水平上调节miR-210。我们的建议在几个方面是新颖和独特的：1）它将研究miR-210在缺血性心脏病中的作用，2）它将使用涉及氧化还原敏感探针的新方法来测量细胞ROS水平，3）它将评估miR-210对心肌细胞中HIF介导的应答的相对贡献，4）将研究一种新的方法，使用金结合的纳米颗粒将miR-210沉默分子引入心肌细胞，5）将使用miR-210和VHK KO小鼠。该提案基于调查人员之间的强有力合作，他们是各自领域的专家，每个人都为项目带来了完全不同的技能。这些研究有望推进我们对缺血诱导细胞死亡的分子基础的认识。

DESCRIPTION (provided by applicant): Ischemic heart disease (IHD) is a major epidemic throughout the developed world, however, the molecular mechanisms that lead to myocardial cell death in IHD are not totally understood. microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to their target mRNA and causing degradation or translational inhibition. We recently showed that the levels of miRNA-210 (miR-210) are elevated in cardiomyocytes exposed to hypoxia. miR-210 response to hypoxia is attenuated in p53-/- mouse embryonic fibroblasts (MEF), implicating a role for p53 in the regulation of miR-210. However, it is not known what role miR-210 plays in cardiomyocyte response to hypoxia. Furthermore, although both miR-210 and hypoxia- inducible factor (HIF) are activated by hypoxia and HIF regulates miR-210 expression, the relative contribution of miR-210 to the biological effects of HIF activation is not known. In this proposal, we will elucidate the mechanism of miR-210 regulation by hypoxia and will determine the role of miR-210 in cardiomyocyte response to ischemia-reperfusion (I/R). Our central hypothesis is that miR-210 is regulated by p53 at the transcriptional level, reduces cardiomyocyte death in response to I/R, and mediates some of the physiological effects of HIF. In Aim 1, we will determine whether miR-210 plays a role in cardiomyocyte cell death and ROS production in response to I/R. In addition to in vitro studies, we will also study miR-210 KO mice and mice with cardiac miR-210 knock down using a novel approach with gold conjugated nanoparticles. We will subject these mice to I/R followed by measurement of cell death and ischemic damage. In Aim 2, we will determine the role of miR-210 in the biological effects of HIF activation. We will overexpress miR-210 in HIF11-/- MEFs, followed by measurement of various physiological parameters in response to hypoxia. We will also use cardiac specific Von Hippel-Lindau (VHL) knockout mice that have constitutively active HIF and display several abnormalities including atrial tumor formation, lipid accumulation and cardiomyopathy. We will cross VHL-/- mice with miR-210 knockout animals and will assess whether miR-210 knockout alters changes from chronic HIF activation in the heart. Finally, in Aim 3, we will elucidate the mechanism of miR-210 regulation by p53. We will assess whether p53 regulates miR-210 at the transcriptional level by measuring miR-210 precursor molecules in p53-/- MEFs and performing ChIP and nuclear run-on assays. Our proposal is novel and unique in several ways: 1) it will study the role of miR-210 in ischemic heart disease, 2) it will use a novel approach involving redox-sensitive probes to measure cellular ROS levels, 3) it will evaluate the relative contribution of miR-210 to HIF-mediated response in cardiomyocytes, 4) it will study a novel approach to introduce miR-210 silencing molecules into cardiomyocytes using gold-conjugated nanoparticles, and 5) it will use miR-210 and VHK KO mice. The proposal is based on a robust collaboration among investigators who are experts in their fields and each bring an entirely different skill set to the project. These lines of investigation promise to advance our knowledge of the molecular basis of ischemic-induced cell death.