【摘要】 目的：探究miR-15b介导PINK1/Parkin线粒体自噬通道在心力衰竭过程中的作用机制，寻找防治心衰的新靶点。方法：通过高通量测序检测心衰患者与健康志愿者体内表达差异micro RNA；体外实验培养HL-1心肌细胞，通过Ang II诱导心肌细胞损伤模型，采用细胞转染技术过表达和抑制miR-15b水平，利用q RT-PCR检测miR-15b转染效能及在各组的表达水平；使用双荧光素酶报告基因实验验证miR-15b与PINK1靶标关系；运用Western Blot检测自噬蛋白Beclin-1、LC3B及线粒体自噬通道蛋白PINK1、Parkin等表达水平；运用免疫荧光法检测心肌细胞PINK1、Parkin蛋白表达情况。结果：心衰患者和健康志愿者外周血中micro RNA表达存在明显差异，其中心衰患者外周血清miR-15b水平上调明显[log2(Fold＿change)>1; P<0.01]，通过对miR-15b靶基因进行预测，发现PINK1是其靶标，并通过双荧光素酶报告基因实验进一步确定miR-15b与PINK1之间的靶向关系。细胞实验中，与空白组相比，模型组miR-15b的表达增多（P<0.05），自噬蛋白LC3B、Beclin-1水平下降（P<0.01），线粒体自噬通道蛋白PINK1、Parkin水平下降（P<0.01）；与模型组相比，转染miR-15b mimics后上述趋势显著增强（P<0.05）；转染miR-15b inhibitor后可逆转上述趋势（P<0.01）。结论：miR-15b可通过介导PINK1/Parkin线粒体自噬水平信号通道调控自噬水平，可能成为心力衰竭潜在的治疗靶点。更多还原

【Abstract】 Objective:To explore the mechanism of miR-15b-mediated PINK1/Parkin mitophagy pathway in the process of heart failure(HF) and to find a new target for prevention and treatment of HF.Methods:The differential expression of microRNA between patients with HF and healthy volunteers was detected by High-throughput sequencing;HL-1 cardiomyocytes were cultured in vitro,cardiomyocyte injury model was induced by Ang Ⅱ,overexpression and inhibition of miR-15b by cell transfection technique,the transfection efficiency of miR-15b and the expression level of miR-15b in each group were detected by qRT-PCR,and the targeting relationship between miR-15b and PINK1 was verified by double luciferase reporter gene experiment.Western Blot was used to detect the expression of autophagy protein Beclin-1,LC3B and mitophagy protein PINK1 and Parkin,and immunofluorescence was used to detect the expression of PINK1 and Parkin protein in cardiomyocytes.Results:There were significant differences in the expression of microRNA between patients with HF and healthy volunteers.The level of miR-15b in patients with HF was significantly upregulated [log2(Fold_ change)> 1;P<0.01].By predicting the target gene of miR-15b,it was found that PINK1 was the target,and the targeting relationship between miR-15b and PINK1 was further determined by double luciferase reporter gene.In the cell experiment,compared with the blank group,the expression of miR-15b in the model group increased(P<0.05),the levels of autophagy LC3B and Beclin-1 decreased(P<0.01),and the level of mitophagy signal pathway PINK1 and Parkin decreased(P<0.01).Compared with the model group,the above trend was significantly enhanced after transfection of miR-15b mimics(P<0.05),and could be reversed after transfection of miR-15b inhibitor(P<0.01).Conclusion:miR-15b can regulate the level of autophagy by mediating the signal pathway of PINK1/Parkin mitophagy,which may be a potential therapeutic target for HF.更多还原