ciRs-MFACR/miR-30a相互作用调控GATA-5在衰老心肌缺血后处理自噬水平降低中的机制研究

The decrease of autophagy level in aged myocardium is an important mechanism of ischemic post-conditioning (IPO) against I/R injury. Circular RNA (ciRs) and miRNA (miRs) are similar in regulation mode and ciRs is rich in miRNA binding sites, so the interaction between ciRs and miRs can counteract the inhibition of the target gene, but the mechanism is unclear. GATA-5 can specifically combine the highly conserved DNA sequence of the target gene, which is the key gene for regulating the function of myocardium. Therefore, it is speculated that the interaction between ciRs and miRs mediates GATA-5-induced the autophagy level decreased, which is an important mechanism for IPO against I/R in aged myocardium. To verify this hypothesis, ciRs-MFACR and miR-30a were screened and identified as specific ciRs and miRs of decreased autophagy level of IPO in aged myocardium. By using immune coprecipitation and biotin specific probe respectively precipitate or pull-down the complex that combinated with ciRs-MFACR and miR-30a, which to clarify the mechanism of interaction between them; Using GATA-5 antibody to carry out immune RNA coprecipitation and mass spectrometry analysis, which to confirm that GATA-5 is a functional target gene of ciRs-MFACR and to reveal the mechanism of the interaction between ciRs-MFACR and miR-30a in decreased autophagy levels of IPO in aged myocardium. Exploring key targets and potential drug target for aging myocardium against I/R injury.

衰老心肌自噬水平降低是缺血后处理(IPO)抗缺血/再灌注(I/R)损伤的重要机制。ciRs和miRs在调节方式上具有相似性，且ciRs富含miRs结合位点，二者可相互作用抵消对靶基因的抑制，但机制未清。GATA-5能特异性结合靶基因高度保守的DNA序列，是调控心肌功能的关键基因，故推测：ciRs和miRs相互作用介导GATA-5引起自噬水平降低是衰老心肌IPO抗I/R损伤的重要机制。为了验证该假说，筛选并确定ciRs-MFACR和miR-30a是衰老心肌IPO自噬水平下降的特异性ciRs和miRs；采用免疫共沉淀和生物素特异性探针分别沉淀或下拉与两者结合的复合物，明确两者相互作用的机制；利用GATA-5抗体进行RNA免疫共沉淀及质谱分析，确定GATA-5是ciRs-MFACR功能性靶基因，旨在揭示两者相互作用在衰老心肌自噬水平降低的机制，探寻关键靶点，为衰老心肌抗I/R损伤提供潜在药靶。

背景：自噬是进化过程中形成的一种自我保护机制，参与了缺血后处理（IPostC）对衰老心肌缺血/再灌注（I/R）损伤的保护作用，但机制不清。ciRs和miRs在调节方式上具有相似性，且ciRs富含miRs结合位点，二者可相互作用抵消对靶基因的抑制，但是否参与IPostC的保护作用尚不清楚。.内容：通过复制衰老心肌IPostC模型，高通量RNA测序等技术筛选并确定衰老心肌IPostC自噬水平下降的特异性ciRs(提示为ciRs-MFACR)；敲除和过表达ciRs-MFACR后检测衰老心肌自噬水平，明确ciRs-MFACR在衰老心肌IPostC自噬水平下降中的作用；生物信息学分析预测能与ciRs-MFACR相互作用的miRs，并用qRT-PCR和双荧光素酶系统验证(提示为miR-30a)；同时检测ciRs-MFACR与miR-30a的表达和定位及对自噬的影响；此外，通过生物信息学分析、双荧光素酶检测及亚硫酸氢盐测序(BSP)等明确HpostC通过Dnmt3b介导miR-30a启动子DNA低甲基化导致miR-30a表达升高。生物信息学分析预测miR-30a的功能靶基因（提示为GATA-5），并用双荧光素酶报告系统验证二者的靶向调控关系；干扰和过表达GATA-5后检测衰老心肌细胞自噬水平和细胞损伤情况，明确miR-30a通过靶向抑制GATA-5调控衰老心肌细胞自噬。.结果：首先，通过对自噬相关指标的检测发现衰老心肌自噬水平降低是IPostC保护作用的重要机制；RNA测序结果显示ciRs-MFACR是衰老心肌IPostC抑制自噬的特异性ciRs，在IPostC衰老心肌组织中表达降低。同时ciRs-MFACR作为miR-30a的“海绵”，与miR-30a相互作用调控衰老心肌细胞自噬水平。HpostC还可以通过Dnmt3b介导miR-30a启动子DNA低甲基化导致miR-30a在衰老心肌HPostC中表达升高。GATA-5是miR-30a的功能靶基因，在衰老心肌I/R损伤中表达降低，过表达后可以缓解衰老心肌I/R损伤后的自噬水平和细胞损伤。.该项目已见刊发表SCI论文3篇，中文核心期刊4篇，专利1项；培养博士研究生2人、硕士研究生3人。.意义：本课题的实施将有利于阐明衰老心肌IPostC的分子机制，确定关键靶点，为衰老心肌I/R损伤的靶向治疗提供新的干预途径。