miRNAs在衰老心肌缺血后处理自噬水平降低中的作用及表观遗传学调控机制

Due to the perfect protection ischemic postconditioning (IPO) has become a hot spot of current study on the resistance of ischemia reperfusion injury ,Our preliminary studies found that the decrease of the level of autophagy is closely related to the protection of aging myocardial ischemic postconditioning (IPO), while, the exact mechanisms of which are still unclear.In addition to that, miRNAs are involved in the regulation of autophagy, and histone acetylation could regulate gene expression in coordination with DNA methylation .However, which means the miRNAs decrease the level of autophagy in aging myocardial ischemic postconditioning is remaining for further study. On the basis of screening differences between miRNAs in aging myocardia, the purpose in this project is to construct the vector include its inhibitor, overexpression and gene related to autophagy , then transfect to clarify its targeted macro-control mechanism. Bi-directional regulation from the miR-133 promoter region of DNA methylation and histone acetylationnbsp; is adopted to explore the influence of the other party on DNA/histone acetylation after the mode of their balance being broken，to elucidate its regulatory mechanism；To analyse the change of miR-133 via the co transfection of sudtained activaton/inactivation mutant of c-myc with the plasmid of DNMT1 and/or HDACS, then to reveal the mechanism that histone acetylation and DNA methylation regulate the change of autophagy induced by miR-133 in collaboration with c-myc and miR-133 .In this way, we could provide the potential drug targets for preventing ischemic reperfusion injury (I/R) of aging myocardial.

缺血后处理(IPO)因其良好的保护作用成为抗缺血/再灌注(I/R)损伤研究的热点，前期研究发现细胞自噬水平降低与衰老心肌IPO密切相关，而miRNAs参与了细胞自噬的调控，且组蛋白乙酰化和DNA甲基化具有协同转录因子调控基因表达的作用，但其在衰老心肌IPO自噬水平降低中的作用机制尚未清楚。本研究拟在筛选和确定衰老心肌IPO差异性miRNAs(miR-133)的基础上，构建其抑制物、过表达及携载自噬关键基因载体并转染，明确其作用机制；从miR-133启动子区DNA甲基化和组蛋白乙酰化双向调控入手，探讨各自平衡模式被打破后，另一方对DNA/组蛋白乙酰化的影响；将c-myc持续失活/激活突变体与DNMT1和/或HDACs质粒共转染，分析miR-133表达的变化，揭示组蛋白乙酰化和DNA甲基化相互作用协同c-myc调控miR-133致细胞自噬水平降低的机制，为衰老心肌I/R损伤的防治提供潜在药靶。

背景：缺血/再灌注(I/R)损伤是心梗病人从再灌注治疗中获得最佳疗效的主要难题。缺血后处理(IPostC)因其良好的保护作用成为抗I/R损伤研究的热点，前期研究发现细胞自噬水平降低与衰老心肌IPostC密切相关，而miRNAs参与了细胞自噬的调控，但其在衰老心肌IPostC自噬水平降低中的作用机制尚未清楚。.内容：复制衰老心肌IPostC及衰老心肌细胞HPostC模型，筛选差异性miRNAs(miR-30a)并验证；构建其抑制物、过表达及携载自噬关键基因载体并转染，明确其作用机制；从miR-30a启动子区DNA甲基化和组蛋白乙酰化双向调控入手，探讨各自平衡模式被打破后，另一方对DNA/组蛋白乙酰化的影响；将DNMT3b或HDAC2单独或同时干扰后，分析miR-30a表达的变化及ChIP检测c-Myc在miR-30a启动子区上的结合情况，揭示组蛋白乙酰化和DNA甲基化相互作用协同c-myc调控miR-30a致细胞自噬水平降低的机制。.结果：RNA测序技术筛选衰老心肌细胞中特异性miR-30a，并采用q-PCR进行验证发现；与Normoxia相比，H/R 条件下miR-30a 表达水平降低，与 HR 相比，HPostC组miR-30a水平升高；荧光素酶报告基因系统证实miR-30a可靶向调控BECN1；单独干扰DNMT3b或HDAC2时，miR-30a的表达升高且c-Myc在miR-30a启动子区的结合增加；与各自单独干扰时比较，将DNMT3b和HDAC2同时干扰后miR-30a的表达升高及c-Myc在miR-30a启动子区上的结合增加更明显。.该项目共发表中文期刊3篇,1篇SCI。.意义：该课题的实施有利于阐明衰老心肌 IPostC 的分子机制，寻找致病环节，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，为衰老心肌 I/R损伤这一全球重大疾病的防治工作提供更多研究资料。