miR-1靶向HSP90调节心肌细胞凋亡影响心肌缺血再灌注损伤的分子机制研究

Myocardial reperfusion therapy remained the most effective way to save the ischemic dying myocardium, but it can also lead to myocardial ischemia-reperfusion injury, with the exact mechanism largely unknown. Our previous studies found that the level of serum miRNA-1 was positively correlated to the myocardial ischemia-reperfusion injury. Recent studies found that HSP90, as a chaperone protein, involved in regulating myocardial apoptosis in myocardial ischemia- reperfusion injury. The bioinformatics analysis showed that HSP90 may be the target genes of miR-1. This project intends to carry out the following studies based on the previous findings on myocardial cell H9C2: (1) Verifying the regulating effect of miR-1 targeting HSP90 through experiments such as luciferase activity analysis; (2) Changing the expression of miRNA-1 to analyze its regulation effect on HSP90 in myocardial apoptosis; (3)Analyzing the HSP90 molecular networks involving in the regulation of myocardial apoptosis on the levels of mRNA-1 and proteins by cDNA microarray and proteomic analysis; (4) Investigating the animal models and the patients' blood samples to further improve the results of cell experiments. Hereby, the study enriches the molecular mechanism of myocardial apoptosis in myocardial ischemia-reperfusion injury, serving as the scientific basis for ongoing study of myocardial ischemia-reperfusion injury involving miRNA-1 and HSP90.

再灌注治疗能挽救缺血濒死心肌同时可引发缺血再灌注损伤,其确切机制尚未明确。本课题组前期研究表明血清miRNA-1水平和缺血再灌注损伤程度成正比，已知HSP90作为伴侣蛋白参与调节心肌缺血再灌注损伤中细胞凋亡，生物信息学分析HSP90可能是 miR-1 的靶基因。本课题拟在先期研究的基础上对心肌细胞H9C2开展以下研究：（1）利用荧光素酶活性分析等实验验证miRNA-1对HSP90 表达的靶向调控作用;（2）基因转染增加或降低细胞中miRNA-1 的表达，分析其调控HSP90 表达对心肌细胞凋亡的影响；(3)基因芯片和蛋白质组学技术从mRNA和蛋白水平分析HSP90 参与心肌细胞凋亡调控的分子网络；(4)利用动物模型和患者血液标进一步验证细胞实验的结果。从而进一步丰富心肌细胞缺血再灌注损伤中细胞凋亡的分子机制，为下一步开展基于miRNA-1和HSP90逆转心肌细胞缺血再灌注损伤的研究提供依据

再灌注治疗在救治急性心肌梗死后濒死心肌的同时可引发缺血再灌注损伤，其确切机制尚未明确。既往研究提示mir-1水平与再灌注损伤相关。生物信息学分析提示Hsp90可能是miR-1的靶向调控基因参与心肌缺血再灌注损伤的调控。课题在利用荧光素酶报告实验验证miR-1对Hsp90表达存在转录后调控作用的基础上，观察miR-1，Hsp90及凋亡相关蛋白分别在急性缺血再灌注及延迟缺血再灌注损伤动物及细胞模型上的表达及分布情况。利用转染技术及siRNA技术分别上调miR-1与HSP90表达，分析其调控心肌细胞发生凋亡的影响。根据心脏超声及流式细胞学检查结果探索急性及延迟缺血再灌注的SD大鼠及心肌细胞模型。心脏缺血再灌注模型以前降支结扎及开通再灌完成，心肌细胞采用低氧/复氧操作模拟缺血再灌注损伤。SD大鼠急性缺血再灌注模型缺血时间为1小时(Ischemia)，再灌注(Reperfusion)24-72小时；延迟再灌注模型为I 4h / R 24h；急性I/R模型心肌细胞给予 I 4h/ 12h,延迟再灌注组;同期入选心肌梗死再灌注患者，记录心脏大体超声表现，比较模型模拟效果并检测相关基因及蛋白的表达情况。原代细胞给予I 4h / R 12h处理模拟急性缺血再灌注，给予I 24h / R 12h处理模拟延迟再灌注损伤。急性再灌注动物模型中，miR-1表达下降30-60%，Hsp90基因表达下调14-40%；蛋白转录上调48-85%，模型组凋亡蛋白caspase-3蛋白活化上调33-210%；延迟缺血再灌注动物模型miR-1表达下降32-35%，Hsp90基因表达下调14-28%；蛋白转录上调39-54%，模型组凋亡蛋白caspase-3蛋白活化上调56-170%。腺病毒转染上调miR-1，miR-1表达上调1000倍，Hsp90表达下调至50%,凋亡蛋白表达上调，Bax/bcl-2 值增加，活化Caspase-3增加100%，凋亡比例增加。腺病毒转染miR-1 inhabitor后，miR-1表达下调50倍，Hsp90表达上调50%，凋亡率下降。课题不仅展示miR-1及Hsp90蛋白在动物及细胞的表型变化，且阐明了miR-1与Hsp90蛋白转录后靶向调控作用，证实miR-1/Hsp90轴在缺血再灌注损伤时心肌细胞调控凋亡机制。