鞘氨醇磷酸胆碱促进缺氧心肌线粒体自噬的分子机制研究

Mitophagy is important in protecting cells against apoptosis by removal of damaged mitochondria. It also is a new target for treatment of myocardial ischemia. So far, the mechanism of transcriptional regulation in mitophagy is not yet clear. We found that sphingosylphosphorylcholine (SPC) was a noval mitophagy inducer. Our research about NR4A2 showed that it promoted autophagy and inhibited apoptosis. Furthermore, NR4A2 was controlled by SPC. We deduced that NR4A2 might be a key factor in SPC-promoted mitophagy. SPC also elevated the mitophagy adaptor protein optineurin (OPTN) and reduced miR-206-3p, miR-455-5p, miR-212-3p. Based on our results and the software prediction, we predicted that SPC promoted NR4A2 transcription by inhibiting microRNAs including miR206-3p, miR455-5p and miR212-3p, and then NR4A2 upregulated the transcription of OPTN, thus causing mitophagy. Gene regulation technology including function blocking and gene overexpression was used as a basic method. We aimed to verify the role of NR4A2 in SPC-promoted mitophagy firstly. Then ChIP combined with locus mutation assay were used together to verify the transcription regulation and regulatory sites of NR4A2 on OPTN. The dual luciferase report assay was used to verify that NR4A2 is the target of microRNA mentioned above. Lastly, we will clarify the role of microRNA /NR4A2/ OPTN pathway in acute myocardial infarction by using the mouse acute myocardial infarction model, so as to provide new experimental evidence to elucidate the mechanism of transcriptional regulation in mitophagy, and provide new targets for the prevention of myocardial ischemia diseases.

线粒体自噬是抑制心肌凋亡的重要方式，也是心梗治疗的新靶标，但其转录调控机制不明确。我们发现鞘氨醇磷酸胆碱（SPC）能促进缺氧心肌线粒体自噬；且NR4A2促进自噬抑制凋亡，受到SPC调控，可能是SPC促自噬的关键因子；该过程伴随自噬衔接蛋白OPTN 的升高及miR206-3p，miR455-5p，miR212-3p的降低。基于预实验及软件预测，我们假设SPC解除了上述miRNA对NR4A2的转录抑制，促进了NR4A2对OPTN的转录调控，促进线粒体自噬。本项目拟以基因干预为基础，证明NR4A2在SPC促自噬中的作用；结合ChIP，突变等研究NR4A2对OPTN的调控及位点，并寻找NR4A2新的靶基因；结合双荧光素报告基因研究miRNA对NR4A2的靶向。利用小鼠模型研究miRNA/NR4A2/OPTN通路在心梗中的作用。为阐明线粒体自噬的机理提供新的实验证据，为心梗防治提供新靶点。

线粒体稳态维持对于心肌细胞的存活具有重要意义。线粒体自噬是影响线粒体稳态的重要机制之一，成为心梗治疗的新靶标，但其转录调控机制不明确。本项目在前期发现SPC能够促进自噬的基础上，进一步研究了SPC调控线粒体自噬的分子机制，发现SPC能通过抑制miR212-3p，解除miR212-3p对NR4A2的转录抑制，促进了NR4A2对线粒体自噬的受体蛋白OPTN的转录调控，促进线粒体自噬。本项目首先以基因干预为基础，证明了NR4A2在SPC促自噬中的作用；结合双荧光素报告基因研究了miRNA对NR4A2的靶向调控，发现miR212-3p对NR4A2的靶向抑制作用；研究了NR4A2通过OPTN参与自噬的信号通路；利用小鼠模型研究了NR4A2在心梗中的保护作用。为阐明线粒体自噬的机理提供新的实验证据，为心梗防治提供新靶点。利用SPC诱导线粒体自噬的细胞模型结合蛋白水平磷酸化组学分析及转录组分析结果，找到了MORN4，EGR1，GATAD1，EPAS1等参与自噬的新因子，并发现了β环糊精等新的自噬诱导剂，为下一步的工作开展打下基础。