慢性心衰中miRNA-199a促进心肌钙离子超载的功能与机制研究

Heart failure remains one of the leading causes of mortality and morbidity in China. However, it remains unclear about the molecular mechanism of heart failure. Recently, we have reported that calcium-overload in cardiomyocytes played an essential role in heart failure. However, the molecular mechanism about calcium-overload in heart failure remains to be identified. As reported, dysregulation of SERCA2 was markedly involved in calcium-overload in heart failure. Interestingly, we have found that miR-199a in neonatal cardiomyocytes could markedly decrease the expression of SERCA2. We proposed that up-regulation of miR-199a in cardiomyocytes might promote calcium-overload by decreasing the expression of SERCA2. In this project, we would verify this hypothesis. Firstly, we would perform the in vivo transfection of miR-199a into cardiomyocytes of rat using adenovirus, and then using lengendorrf system to isolate the cardiomyocyte of the left ventricular. Using microscopy, we could pick the GFP-positive cardiomyocytes to perform confocal and patch-clamp analysis to see whether over-expression of miR-199a could aggratate the calcium overload in cardiomyocytes. Secondly, we would establish a calcium-sensitive fusing protein, GCaMP2, transgenic zebrafish for in vivo calcium examination. Then over-expression or knock-down of miR-199a would be performed in these transgenic zebrafishes to clarity that miR-199a could promote calcium-overload in heart failure. Thirdly, we would establish miR-199a transgenic zebrafish to find that miR-199a could significantly promote the process of heart failure. Lastly, we would use Ago2 co-immunoprecipitation-microarray to identify the potential targets of miR-199a, then we would use luciferase-Western blot to verify these targets of miR-199a. This project would provide new knowledge about miRNA in heart failure.

慢性心衰的发病率和死亡率逐年升高。心肌钙超载在慢性心衰中发挥重要作用。我们前期研究发现在慢性心衰中，肌浆网钙泵SERCA2表达降低，miR-199a表达升高；过表达miR-199a能够抑制心肌细胞SERCA2的表达。由于SERCA2表达降低是诱发心肌细胞钙超载的重要因素，因此我们推测：miR-199a能够通过抑制SERCA2表达，加剧心肌钙超载，促进心衰的进展。本课题拟通过大鼠在体过表达miR-199a，激光共聚焦-膜片钳检测心肌细胞钙离子、钙离子指示剂GCaMP2转基因斑马鱼-激光共聚焦整体心脏检测钙离子的方法，明确miR-199a在心力衰竭钙超载中的作用。2. 采用Luciferase-免疫印迹技术验证SERCA2为miR-199a的靶基因，通过Ago2免疫共沉淀-基因芯片筛选miR-199a其它靶基因，明确miR-199a作用的分子机制，为心力衰竭的治疗提供理论基础。

本课题完成了项目书的研究目标。我们确定了miR-199a在生理状态和病理状态下，通过靶向抑制HSPA5的蛋白表达对营养剥夺诱发的乳鼠心肌细胞自噬具有促进作用。我们发现miR-199与miR-214同时被转录在DNM3OS前体RNA中。我们构建了miR-199、miR-214和DNM3OS的敲除大鼠。我们发现miR-199、miR-214敲除大鼠发育正常，但DNM3OS敲除影响大鼠整体发育。我们对心肌肥厚大鼠模型进行miR-199、miR-214的表达检测，结果显示miR-199和miR-214随着肥厚程度的加重，表达逐渐升高。我们对miR-199敲除和miR-214敲除大鼠进行功能研究，显示敲除鼠和野生鼠在心肌厚度上没有差别，然而钙离子通道蛋白CACNA1c和肌浆网钙泵ATP2a2在miR-199敲除大鼠中显著降低，PLN在miR-199敲除大鼠中磷酸化显著升高，这些结果表明miR-199对肌浆网钙离子具有重要调控作用。我们进一步通过主动脉缩窄模型对miR-199敲除大鼠进行心肌肥厚相关研究，结果显示miR-199敲除促进了心肌肥厚的发生。这一心肌肥厚的发生可能与miR-199调控钙离子相关蛋白、线粒体相关基因、心肌纤维化和血管新生有关。我们在冠状动脉左前降支结扎导致的心肌缺血模型中，发现miR-199表达明显降低。我们检测了miR-199敲除的心脏功能在LAD结扎后，梗死面积明显增加，纤维化减弱。我们通过RNA测序系统性筛选受miR-199敲除影响的基因，发现升高的基因主要集中在细胞周期，降低的基因主要集中在炎症反应。我们通过生物信息学分析miR-199的潜在靶基因，并通过双荧光素酶报告基因加以验证，结果显示CDK7，YAP1等的3’非编码区能够被miR-199所识别和抑制，提示了miR-199可能通过调控这些基因的表达发挥生物学功能。本课题的相关研究结果发表高水平研究论文6篇，其中IF>5的文章2篇，培养博士后1名，博士5名，硕士2名。