miR-100介导自噬调控自发性高血压大鼠左室肥厚的实验研究

Left ventricular hypertrophy is a common characteristic change among hypertensive heart damages, and has become an independent risk factor for many cardiovascular events. Mechanism study is important to design strategies to delay or reverse left ventricular hypertrophy and thus prevent hypertension. Our preliminary studies have shown that miR-100 was highly expressed in the peripheral circulation of patients with essential hypertensive left ventricular hypertrophy. Meanwhile, the literatures have reported that both miR-100 and autophagy were involved in the process of cardiac hypertrophy, and miR-100 played an important role to regulate autophgy. Based on our preliminary study and the literature reports, we hypothesize that miR-100 and its downstream target genes regulate autophagy and induce hypertensive left ventricular hypertrophy. We propose to screen the autophagy associated-downstream genes of miR-100 by bioinformatics analysis and expression examination in the hypertrophy myocardium of spontaneously hypertensive rats(SHRs). We will examine the effects of miR-100 in left ventricular hypertrophy, its downstream target genes and autophagy through transfecting recombinant lentivirus containing miR-100 counter into SHRs, whose left ventricle was confirmed to be hypertrophy. Target genes of miR-100 will be verified by dual luciferase gene reporter assay.We will modulate the levels of miR-100, its downstream target genes and autophagy in hypertrophic cardiomyocyte model by recombinant lentivirus technique and autophagy drugs, and further examine the mechanism of miR-100 and its downstream target gene-mediated autophgy in hypertensive left ventricular hypertrophy. We expect to reveal the role and mechanism of miR-100 in hypertensive left ventricular hypertrophy, and provide the rational to design new therapeutic strategies to prevent and treat hypertensive left ventricular hypertrophy.

左室肥厚是高血压心脏损伤最常见的特征性病理变化，是多种心血管事件的独立危险因素，探索其发生机制有助于高血压防治。我们前期研究发现，miR-100高表达于原发性高血压左室肥厚患者外周循环；现有文献表明，miR-100、自噬均参与心肌肥厚病理过程，且miR-100参与调控自噬。由此我们假设：miR-100作用相关靶基因介导自噬参与高血压左室肥厚的发生。本研究拟通过生信分析及SHRs肥厚心肌检测，筛选出miR-100自噬相关靶基因；miR-100反向干预慢病毒活体转染左室肥厚SHRs，观察miR-100对左室肥厚、靶基因及自噬活性的影响；双荧光素酶基因报告技术验证miR-100靶基因；重组慢病毒技术、自噬活性药物组合干预肥大心肌细胞，分析miR-100作用靶基因介导自噬参与高血压左室肥厚发生的机制。研究的实施将有助于阐明miR-100对高血压左室肥厚的作用及调控机制，并可能为其防治提供新的靶点。

左室肥厚是高血压心脏损伤最常见的特征性病理变化，是多种心血管事件的独立危险因素，探索其发生机制有助于高血压防治。基于自噬在心肌肥大中发挥重要作用的科学证据，本研究旨在探讨 miR-100-5p 与自噬在心脏肥大发展中的关系。根据miR-100-5p阶段性高表达于自发性高血压大鼠（SHR）肥厚心肌及血管紧张素II（Ang II）诱导的心肌细胞肥大模型中，本项目设计一系列体外和体内实验以探索miR-100-5p对自噬和心脏肥大的作用。通过双荧光素酶报告基因技术、RNA免疫沉淀 (RIP)、定量实时PCR (qRT-PCR)、蛋白质印迹、免疫荧光和透射电子显微镜 (TEM) 等方法探索miR-100-5p 介导自噬对心肌肥大的调控机制。结果发现miR-100-5p在肥大心脏和Ang II诱导的心肌细胞中高表达；miR-100-5p的过表达促进了心肌肥大标志物ANP、BNP和β-MHC的表达并增加心肌细胞表面积，而miR-100-5p抑制剂下调miR-100-5p表达则可抑制上述生物学效应，下调miR-100-5p 表达可显着改善心脏功能并减轻SHR左室肥厚；体外机制研究发现，miR-100-5p 通过靶向 mTOR 促进心肌细胞自噬，自噬抑制剂3-甲基腺嘌呤 (3-MA) 或 mTOR 过表达可通过抑制自噬部分逆转 miR-100-5p 促心肌细胞肥大效应。结论：本研究阐明miR-100-5p通过靶向mTOR介导自噬激活促进心肌肥大,此研究成果可能为高血压左室肥厚的防治提供新的靶点，助力高血压防治技术的创新发展。