Oip5-as1作为“分子脚手架”抑制心肌缺血再灌注损伤中线粒体异常分裂的机制研究

线粒体异常分裂是心肌缺血再灌注（MI/R）损伤发生的关键，但其分子机制尚不明晰。本课题组前期研究发现lncRNA Oip5-as1在MI/R中低表达。应用CRISPR/dCas9 SAM技术过表达Oip5-as1可抑制线粒体过度分裂，从而缓解MI/R进程中线粒体途径的细胞凋亡。这提示Oip5-as1是线粒体分裂的重要调控分子。进一步借助生信分析工具和细胞实验，我们推测出Oip5-as1绑定PIM1激酶与DRP1形成“分子脚手架”结构，使得DRP1在S637位点的磷酸化增加，进而抑制DRP1向线粒体转位及其介导的线粒体异常分裂。为此，本项目将设计过表达、敲减及挽救实验明确Oip5-as1对线粒体形态功能的影响。并利用RNA pull-down、RIP、Co-IP、截短与突变实验对其与PIM1、DRP1的交互作用进行精细解析。最后在大鼠模型上加以验证。本课题可为临床防治MI/R损伤提供新靶点。

Abnormal mitochondrial fission plays a key role in the development of myocardial ischemia/reperfusion (MI/R) injury. However, the underlying molecular mechanism is still not clear. Previous study by our group has indicated that the expression level of lncRNA Oip5-as1 was downregulated upon MI/R. Overexpression of Oip5-as1 by CRISPR/dCas9 Synergistic Activation System inhibited excessive mitochondrial fission, and consequently decreased mitochondria-mediated cell apoptosis during MI/R. It was suggested that Oip5-as1 is an important regulatory molecule implicated in mitochondrial fission. Further, by using bioinformatics analysis tools and cell experiments, we proposed that Oip5-as1 promoted the phosphorylation of DRP1 at Ser637 through serving as a scaffold of PIM1 kinase and DRP1. In turn, the phosphorylation of DRP1 prevented translocation of DRP1 to mitochondria and DRP1-dependent fission. To prove the hypothesis, overexpression, knockdown, and rescue experiments are performed to determine the effect of Oip5-as1 on mitochondrial morphology stabilization and function integrity. The RNA pull-down, RIP, Co-IP, truncation test, and mutation experiments are applied to identify the regions of Oip5-as1 that are required for the interaction of PIM1 or DRP1. These effects of Oip5-as1 will also be verified in the MI/R-injured rats. The present study may provide a new therapeutic target for the prevention and treatment of MI/R injury.