circRNA-UXS1.40 - microRNA 146a海绵作用在心肌缺血再灌注损伤中的作用研究

Currently, myocardial ischemia-reperfusion injury is still a major stumbling block to the effect of myocardial ischemia-reperfusion. In our previous study, we found that microRNA 146a increased transiently in the early stage of myocardial ischemia-reperfusion, which could inhibit apoptosis by targeting the gene med1. And after circRNA-UXS1.40 was silenced, which was the possible upstream sponge molecular of microRNA 146a, the protein expression of med1 decreased. Thus we speculated that circRNA-UXS1.40 would aggravate myocardial ischemia-reperfusion injury by its molecular sponge effect to microRNA 146a. If microRNA146a expression was timely regulated, it could protect myocardium. In this project, after confirming whether UXS1.40 is an upstream sponge molecule of microRNA 146a, transfection-experiments and rescue study will be performed in vitro, meanwhile cardiac-specific circ-UXS1.40 overexpression and knockout transgenic mice will be used to establish a model of myocardial ischemia-reperfusion injury in vivo to clarify the effect of circ-UXS1.40 in occurrence and development of myocardial ischemia-reperfusion injury.

心肌缺血再灌注损伤仍是影响心肌缺血再灌注疗效的一大绊脚石。本项目组前期研究发现，心肌缺血再灌注早期代偿性升高的microRNA146a可通过靶作用于med1从而抑制心肌细胞凋亡，而microRNA146a可能的上游海绵分子circRNA-UXS1.40被沉默后，心肌细胞中med1蛋白表达下降。故我们推测circRNA-UXS1.40可能通过其海绵作用影响microRNA146a的作用，进而加重心肌缺血再灌注损伤，若能适时干预，则可保护心肌。本项目在确认circRNA-UXS1.40是否为microRNA146a的海绵分子后，在体外利用转染、补救实验，在体内利用心肌特异性circRNA-UXS1.40基因过表达和敲除小鼠建立在体心肌缺血再灌注损伤模型，最终明确circRNA-UXS1.40-microRNA146a海绵作用对心肌缺血再灌注损伤发生发展的影响。

体内外探究环状RNA Fbxl5是否为microRNA146a的上游海绵分子及Fbxl5通过microRNA146a调控心肌缺血/再灌注损伤的作用及机制研究。方法：我们收集临床急性心肌梗死患者PCI术前及术后不同时间点的外周血，采用RT-PCR方法检测了的表达，发现Fbxl5在再灌注后呈现先升高而后降低的趋势，再通过双荧光素酶报告基因验证Fbxl5与microRNA146a的结合。在体外水平上，采用体外转染p-Lenti- Si- Fbxl5和p-Lenti- Fbxl5，从正负两个效应方面研究心肌细胞在缺氧/复氧干预下，凋亡水平。在体内水平上，构建了小鼠缺血/再灌注损伤模型，通过尾静脉注射AAV9-TNT-Si-CircRNA Fbxl5及AAV9-TNT- CircRNA FBXL5，评估Fbxl5对小鼠经历缺血/再灌注后，心功能、心肌细胞梗死面积、凋亡等指标。结果：1）缺血/再灌注损伤1h后，小鼠血清中Fbxl5水平显著升高；2）我们通过双荧光素酶报告基因验证了Fbxl5作为microRNA的高效海绵分子；3）我们通过腺病毒靶向调控心肌细胞Fbxl5的表达，结果表明：抑制Fbxl5表达后，心功能改善，梗死面积减小，心肌细胞Bcl-2的表达增加，Bax和caspase-3的表达减少；4）在体外研究中，Fbxl5敲除较对照组能减少乳鼠心肌细胞在缺氧/复氧干预下的凋亡水平；5）通过挽救实验，我们明确了Fbxl5通过microRNA146a-med1轴产生调控作用。科学意义: 心肌细胞Fbxl5与调控心肌缺血/再灌注损伤的结局存在相关性。Fbxl5作为一个具有潜在调控意义的位点，具有一定的潜在的治疗靶点作用。