目的:本研究旨在探讨miR-494-3p是否通过抑制PGC1-α的表达来抑制心肌细胞线粒体自噬的发生,为自噬在缺氧/再灌注(H/R)心脏损伤中的作用提供理论基础。
方法:用RT-qPCR检测miR-494-3p的表达,和PGC1-α的表达,自噬相关蛋白(LC3,Beclin1),凋亡相关蛋白(Bax和Bcl-2),免疫印迹法检测PINK1/Parkin信号通路相关蛋白(PINK1、Parkin)和线粒体变化相关蛋白(Mfn1、Mfn2、OPA1)。通过JC-1染色(ΔkWm)检测线粒体膜电位的变化。透射电镜观察自噬体的形成。CCK-8检测细胞增殖活性,流式细胞术检测细胞凋亡。双荧光素酶基因报告基因测定鉴定了miR-494-3p和PGC1-α之间的靶向结合位点。
结果:结果显示miR-494-3p和PGC1-α在H/R心肌细胞中差异表达;即,miR-494-3p的表达下调,PGC1-α的表达上调。此外,H/R心肌细胞发生线粒体自噬。也就是说,LC3-II/LC3-I,Beclin1、PINK1和Parkin表达上调,Mfn1、Mfn2和OPA1表达下调,线粒体膜电位降低。转染miR-494-3p模拟物可以显著提高心肌细胞增殖活性,抑制心肌细胞凋亡和自噬的发生,而miR-494-3p抑制剂的转染则有相反的结果。转染miR-494-3p模拟物后,自噬抑制剂和激活剂治疗改变了miR-494-3p对心肌细胞增殖和凋亡的影响。同时,PGC1-α的过表达逆转了miR-494-3p对心肌细胞增殖的促进作用以及对细胞凋亡和自噬的抑制作用。
结论:MiR-494-3p可以靶向和负调控PGC1-α的表达,从而抑制心肌细胞的线粒体自噬。
The purpose of this study was to explore whether miR-494-3p inhibits the occurrence of mitochondrial autophagy in cardiomyocytes by inhibiting the expression of PGC1-α and to supplement the theoretical basis for the role of autophagy in cardiac injury induced by hypoxia/reperfusion (H/R).
The expression of miR-494-3p was detected by RT‒qPCR, and the expression of PGC1-α, autophagy-related proteins (LC3, Beclin 1), apoptosis-related proteins (Bax and Bcl-2), PINK1/Parkin signaling pathway-related proteins (PINK1, Parkin) and mitochondrial change-related proteins (Mfn1, Mfn2, OPA1) was detected by Western blotting. The changes in mitochondrial membrane potential were detected by JC-1 staining (ΔΨm). The formation of autophagosomes was observed by transmission electron microscopy. Cell proliferation activity was detected by CCK-8, and cell apoptosis was detected by flow cytometry. A dual-luciferase gene reporter assay identified a targeted binding site between miR-494-3p and PGC1-α.
The results showed that miR-494-3p and PGC1-α were differentially expressed in H/R cardiomyocytes; that is, the expression of miR-494-3p was downregulated, and the expression of PGC1-α was upregulated. In addition, mitochondrial autophagy occurred in H/R cardiomyocytes. That is, LC3-II/LC3-I, Beclin 1, PINK1, and Parkin expression was upregulated, Mfn1, Mfn2, and OPA1 expression was downregulated, and the mitochondrial membrane potential was decreased. The transfection of miR-494-3p mimic can significantly improve the cell proliferation activity of cardiomyocytes and inhibit the occurrence of cardiomyocyte apoptosis and autophagy, while the transfection of miR-494-3p inhibitor has the opposite result. After transfection of the miR-494-3p mimic, treatment with autophagy inhibitors and activators changed the effects of miR-494-3p on cardiomyocyte proliferation and apoptosis. At the same time, the overexpression of PGC1-α reversed the promoting effect of miR-494-3p on cardiomyocyte proliferation and the inhibitory effect on apoptosis and autophagy.
MiR-494-3p can target and negatively regulate the expression of PGC1-α to inhibit mitophagy in cardiomyocytes.