Role of miR-574 driver and passenger strands in cardiac hypertrophy

项目摘要： 心血管疾病（CVD）是全球发病率和死亡率的主要原因。更好 了解CVD的病理机制将提高预防和 治疗干预。心血管疾病的一个主要病因是心脏基因表达失调 转录因子（TF）和微小RNA（miRNA）。miRNAs是一种小的非编码RNA 参与基因表达调控。TF通常是较差的小分子药物靶标。 然而，miRNA可以作为独特的治疗剂，因为它们可以靶向多个TF， 相同的病理途径。大多数前体miRNAs（pre-miRNAs）被加工以产生 一种有活性的驱动链;互补的过客链通常被降解。 有趣的是，pre-miR-574产生两个功能链-miR-574- 5 p和miR-574- 3 p。我们发现 miR-574的两条链在保护心脏免受心肌肥大方面发挥协同作用。我们 结果显示，miR-574- 5 p在慢性人心力衰竭组织中相对于正常心脏被诱导。 我们发现，miR-574- 5 p在肥大和缺血应激下上调，并沉默了心肌细胞的凋亡。 三种促肥大TF肌细胞增强因子-2因子（Mef 2a/2c/2d）的表达和 防止心肌细胞（CM）肥大。miR-574- 5 p模拟物在小鼠中的转染 心肌细胞（CM）保护细胞免受异丙肾上腺素（ISO，β-肾上腺素能激动剂）诱导的 肥大和缺氧-血清饥饿诱导的细胞死亡。miR-574敲除小鼠表现出 与纤维化增加和CM增大相关的晚期心脏肥大表型， 与慢性ISO注射后的野生型小鼠相比。我们还发现， miR-574- 3 p与miR-574- 5 p共上调，但被缺氧激活的磷酸化hnRNP捕获 L（异质核核糖核蛋白L）。miR-574- 3 p通过外泌体从CM分泌， 靶向成纤维细胞以减少纤维化。我们的研究目标是解决基本问题， 使用miR-574- 5 p/3 p作为心脏系统中的原型分子的miRNA生物学（差异 规则、机械多样性和驾驶员与乘客之间的功能关系 链），并通过开发靶向TF的基于miRNA的治疗剂来治疗HF。我们的中央 一个假设是：在肥厚和缺血性应激下的CM中， 链miR-574- 5 p和过客链miR-574- 3 p整合心脏基因表达， 调节病理性心脏肥大和重塑。我们将通过以下方式检验这一假设： 追求两个目标。目的1验证miR-574- 5 p调控Mef 2表达的假设 TF和其他心脏基因通过miRISC，从而调节心脏肥大和重塑。 目的2将检验在CM中缺血通过特异性TF促进miR-574转录的假设 并激活P-hnRNP L对miR-574- 3 p的捕获，随后是外泌体分泌， 细胞间通讯

Project Summary: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Better understanding of the pathological mechanisms underlying CVD will improve preventive and therapeutic interventions. One major etiology of CVD is dysregulation of cardiac gene expression by transcription factors (TFs) and microRNAs (miRNAs). miRNAs are small non-coding RNAs involved in gene expression regulation. TFs are usually poor small-molecule drug targets. However, miRNAs can act as unique therapeutic agents because they can target multiple TFs in the same pathological pathway. Most precursor miRNAs (pre-miRNAs) are processed to generate an active driver strand miRNA; the complementary passenger strand is usually degraded. Intriguingly, pre-miR-574 produces two functional strands—miR-574-5p and miR-574-3p. We found that both strands of miR-574 play a synergistic role to protect hearts from cardiac hypertrophy. We showed that miR-574-5p was induced in chronic human heart failure tissues versus normal hearts. We found that miR-574-5p is up-regulated under hypertrophic and ischemic stress and silences the expression of three pro-hypertrophy TFs myocyte enhancer factor–2 factors (Mef2a/2c/2d) and prevents cardiomyocyte (CM) hypertrophy. Transfection of miR-574-5p mimics in mouse cardiomyocytes (CM) protects the cells from isoproterenol (ISO, a β-adrenergic agonist)-induced hypertrophy and hypoxia-serum starvation-induced cell death. miR-574 knockout mice exhibit an advanced cardiac hypertrophy phenotype associated with increased fibrosis and enlarged CM, compared to wild-type mice after chronic ISO injection. We also showed that the passenger strand miR-574-3p is co-upregulated with miR-574-5p but captured by hypoxia-activated phospho-hnRNP L (heterogeneous nuclear ribonucleoprotein L). miR-574-3p is secreted from CM via exosomes and targets fibroblasts to reduce fibrosis. Our research goal is to address fundamental questions in miRNA biology using miR-574-5p/3p as archetypal molecules in the cardiac system (differential regulation, mechanistic diversity, and functional relationship between driver and passenger strands), and to treat HF by developing miRNA-based therapeutics that target TFs. Our central hypothesis is: In CM under hypertrophic and ischemic stress, differential regulation of the driver strand miR-574-5p and passenger strand miR-574-3p integrate cardiac gene expression and modulate pathological cardiac hypertrophy and remodelling. We will test this hypothesis by pursuing two aims. Aim 1 will test the hypothesis that miR-574-5p regulates the expression of Mef2 TFs and other cardiac genes via miRISC, thereby modulates cardiac hypertrophy and remodelling. Aim 2 will test the hypothesis that in CM ischemia promotes miR-574 transcription via specific TFs and activates capture of miR-574-3p by P-hnRNP L followed by exosome secretion and intercellular communication.