九龙藤总黄酮通过lncRNA MIAT/miR-384-5p/Beclin1调控自噬抗急性心肌梗死

Studies have shown that autophagy and non coding RNA play important roles in the occurrence and development of myocardial infarction. Using the bioinformatics database, we predicted that lncRNA MIAT could be combined with miR-384-5p, and Beclin1 gene sequence also has partial base-pairing regions to miR-384-5p. As shown in our previous research, compared with the autophagy inhibitor, 3-MA, pretreatment with Bauhinia championii flavones (BCF) could increase the autophagy level in ischemia period by up-regulating the protein expression of LC3- II and Beclin1 and down-regulating the protein expression of mTOR. On the other hand, BCF attenuated acute myocardial ischemia induced by pituitrin. Our research also revealed that BCF activated the PI3K/ Akt pathway, upregulated the expression of PI3K, inhibited apoptosis and necrosis, and improved the heart function. Above effects of BCF were related to multiple target proteins of autophagy and miR-384-5p. Therefore, we speculated that BCF play the anti-myocardial infarctional effects through mediating lncRNA MIAT/miR-384-5p/Beclin1and regulating autophagy. This project will observe the effects of BCF on myocardial infarction, and further characterize the signaling cascade and specific targets by regulating autophagy through lncRNA MIAT/miR-384-5p/ Beclin1. Multiple technologies will be used, such as cell biology, molecular biology, RNA interference in vivo, animal model, immunology, and so on. These studies will be conducted in vitro and in vivo, including cell, gene and protein levels. According to this project, we hope to provide new ideas on prevention and treatment of myocardial infarction.

研究表明自噬和非编码RNA在心肌梗死的发生发展起重要作用。应用生物信息数据库，我们预测lncRNA MIAT可与miR-384-5p结合，Beclin1基因序列亦有与miR-384-5p碱基配对的区域。课题组研究发现，与自噬抑制剂3-MA相比，九龙藤总黄酮上调缺血心肌LC3-Ⅱ、Beclin1表达，下调mTOR表达，增加缺血期自噬水平；抗垂体后叶素致急性心肌缺血；上调PI3K表达，激活PI3K/Akt通路，抑制细胞凋亡和坏死，改善心功能。上述作用涉及自噬与miR-384-5p的多个靶蛋白，据此推测九龙藤总黄酮通过lncRNA MIAT/miR-384-5p/Beclin1调节自噬，抗心肌梗死。本项目拟通过细胞生物学、分子生物学、体内RNA干扰、疾病动物模型、免疫学等技术，从体内、外层次，细胞、基因和蛋白水平研究九龙藤总黄酮调节自噬，抗心肌梗死的信号级联和具体靶点，为防治心肌梗死提供新思路。

本项目研究分体外体内两部分，应用双荧光素酶报告基因检测、RAP、ELISA、TTC染色、HE染色、透射电镜、激光共聚焦显微镜、RT-qPCR、Western blot等技术，从细胞、分子、整体水平明确九龙藤总黄酮（Bauhinia championii flavones，BCF）通过介导lncRNA MIAT/miR-384-5p/Beclin1调节自噬，抗心肌缺血损伤所依赖的信号级联和具体效应靶点。体外培养H9c2心肌细胞，建立OGD模型，以 MIAT、miR-384-5p、Beclin1为靶点，通过上调或沉默基因，验证lncRNA MIAT、miR-384-5p、Beclin1对缺血心肌细胞的作用，并分析BCF干预前后细胞的变化，明确其对缺血心肌保护的效应靶点。体内建立大鼠AMI模型，应用在体RNA干扰技术，分别沉默lncRNA MIAT和miR-384-5p的基因表达，研究BCF对lncRNA MIAT/miR-384-5p 的影响，对缺血损伤心肌相关生化、病理指标的调节，对自噬相关基因、蛋白的作用，确定九龙藤总黄酮干预减轻急性心肌梗死的体内机制，并验证与体外结果的一致性。.研究结果表明：lncRNA MIAT与miR-384-5p、miR-384-5p与Beclin1之间存在靶向负性调控关系。与模型组相比，BCF预处理显著降低CK-MB、cTnI水平，减少心梗面积，改善心功能；下调MIAT、Atg5，上调miR-384-5p基因表达；降低LC3-II/LC3-I比值，下调Beclin1、Cathepsin D，上调p62基因和蛋白表达，减少自噬通量；降低自噬小体与自噬溶酶体数量，抑制过度自噬（P<0.05或P<0.01），改善心肌缺血损伤，而沉默miR-384-5p则逆转了BCF的上述作用。此外，过表达Beclin 1可促进自噬激活，减弱BCF抑制自噬的功能，而沉默MIAT和过表达miR-384-5p则增强BCF的抑制自噬作用。综上所述，BCF的心肌保护作用与调控lncRNA MIAT/miR-384-5p/Beclin1信号轴，抑制过度自噬有关。.通过本项目研究，阐明九龙藤总黄酮通过lncRNA MIAT/miR-384-5p/Beclin1信号级联，抑制过度自噬，减轻急性心肌梗死的作用及分子机制，为进一步开发防治冠心病新靶点药物提供试验依据。