心肌缺血再灌注过程中,lncRNA-HRIM通过靶向调节miRNA-21-5p干预细胞自噬通路的研究

Excessive autophagy induced cardiac myocytes cell death during ischemia/reperfusion (I/R) period. In this study, we found that lncRNA-HRIM expression significantly upregulated in myocytes subjected to I/R, and is associated with increasing autophagy levels, which lead to enhance cell death. In contrast, miRNA-21-5p expression is significantly downregulated, and miRNA-21-5p mimic in myoctyes contributes to reduce the level of autophagy, eventually decreasing myocardial cell death in response to I/R. These preliminary results tell us that lncRNA-HRIM and miRNA-21-5p play a reverse role in regulating I/R-induced autophagy in cardiac myocytes and effect cardiac I/R injury. Based on it, we will establish a model of I/R injury in cardiac myocytes and investigate the function and mechanism of lncRNA-HRIM and miRNA-21-5p in regulating autophagy pathway of cardiac myocytes injury during I/R period using RNA interference or gene over-expression technology，and then clarify the interaction between lncRNA-HRIM and miRNA-21-5p in cardiomyocytes using luciferase assay and pull-down assay. We try to provide new therapeutic targets for the prevention and treatment of cardiac I/R injury.

在缺血再灌注（I/R）过程中，抑制过度自噬可减少心肌细胞损伤。我们前期发现在I/R过程中，心肌细胞LncRNA-HRIM表达上调，增加自噬促使细胞死亡，而miRNA-21-5p表达下调，它抑制细胞过度自噬，减少心肌细胞死亡。初步结果提示：在心肌I/R过程中，LncRNA-HRIM与miRNA-21-5p可反向调控心肌细胞自噬水平。基于此，我们假设在心肌I/R过程中，lncRNA-HRIM靶向调节miRNA-21-5p，间接调控其靶基因，干预细胞自噬通路。因此，本研究欲构建心肌I/R损伤模型，通过RNA干扰或基因过表达等手段，体内深入探讨LncRNA-HRIM和miRNA-21-5p对心肌细胞自噬水平及心梗面积的影响；同时采用荧光素酶报告基因载体、pull-down等实验阐明在自噬调控层面LncRNA-HRIM和miRNA-21-5p之间的相互作用，为心肌缺血再灌注损伤的防治提供新治疗靶点。

在心肌缺血再灌注过程（I/R）中lncRNA扮演着重要的角色，调控细胞的生存或死亡。心肌细胞过度自噬可增加细胞死亡，且抑制过度自噬可减少心肌细胞损伤。本项目利用microarray芯片筛选出在心肌缺血再灌注过程中差异性表达的lncRNAs，并通过生物信息学分析挑选出lncRNA-HRIM （LncRNA Associated with Hypoxia Reoxygenation Injury in Myocardial cell）进行的PCR表达验证，结果研究发现1)在心肌细胞缺氧再复氧过程中，lncRNA-HRIM显著增加，心肌细胞自噬增加，细胞活力下降，利用siRNA干扰细胞内的lncRNA-HRIM后心肌细胞活性增加，LC3II/I比例下降，p62增加，提示lncRNA-HRIM保护心肌细胞缺血再灌注损伤的机制与下调细胞内过度自噬的活性有关，2)项目证实了miRNA-21-5p在心肌缺氧复氧过程中调控PTEN/Akt/mTOR通路来抑制细胞的过度自噬,减少了心肌损伤,并通过双荧光素酶报告载体揭示了lncRNA-HRIM与miRNA-21-5p之间不存在相互作用，但抑制lncRNA-HRIM可下调心肌miRNA-29a-3p水平； 3）明确了在心肌缺血再灌注过程中心肌细胞焦亡显著增加，而大黄素可抑制lncRNA-HRIM表达、通过调控TLR4/MyD88/NF-κB/NLRP3途径减低GSDMD-N、IL-1β等水平，减少心肌细胞焦亡进而来缓解心肌缺血再灌注损伤，本项目的完成有望为心肌缺血再灌注损伤的防治提供了新的靶点。已发表标注SCI论文8篇，申请发明专利并获授权1项，培养硕士研究生毕业2名。