联合microRNA测序及体外实验探究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  

Combined With MicroRNA Sequencing and In Vitro Experiments to Explore the Mechanism of MiR-15b-ediated PINK mediated PINK1/Parkin Mitophagy Pathway in the process of Chronic Heart Failure

作　　者：皮建彬 王文会[1] 赵华云[1] PI Jian-bin;WAMG Wen-hui;ZHAO Hua-yun(Foshan hospital of TCM,Foshan,Guangdong 528051)

机构地区：[1]佛山市中医院,广东佛山528051

出　　处：《中医康复》2024年第4期11-17,共7页Traditional Chinese Medicine Rehabilitation

基　　金：佛山市科技局医学类科技攻关项目(NO.2220001005032)。

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microRNA;体外实验培养HL-1心肌细胞,通过AngII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qRT-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WesternBlot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microRNA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。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 up regulated[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 miR15b 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 miR15b 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.

关 键 词：心力衰竭 线粒体自噬 miR-15b PINK1/Parkin信号通道 

分 类 号：R364.1[医药卫生—病理学]