miR-202-5p靶向下调SOX6表达对缺氧复氧诱导的心肌细胞氧化应激的影响研究

Effect of miR-202-5p on oxidative stress in cardiomyocytes induced by hypoxia/reoxygenation through down-regulating SOX6

目的:探讨miR-202-5p靶向SOX6对缺氧复氧诱导的心肌细胞氧化应激的影响。方法:体外培养大鼠胚胎心肌细胞H9C2,构建心肌细胞缺氧/复氧损伤模型。将H9C2细胞随机分为空白组、缺氧复氧组、缺氧复氧+miR-con组、缺氧复氧+miR-202-5p组、缺氧复氧+miR-202-5p+pcDNA组、缺氧复氧+miR-202-5p+pcDNA-SOX6组。qRT-PCR和Western blot检测心肌细胞miR-202-5p和SOX6的表达水平。CCK-8法检测心肌细胞存活率。流式细胞术检测心肌细胞凋亡。试剂盒检测活性氧簇(ROS)水平、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)含量变化。双荧光素酶报告基因实验和Western blot验证miR-202-5p和SOX6的靶向调控关系。结果:缺氧复氧处理可显著抑制心肌细胞miR-202-5p表达,促进SOX6表达,提高ROS和MDA水平,降低GSH-Px水平,抑制细胞存活,促进细胞凋亡。上调miR-202-5p表达可显著降低ROS和MDA水平,提高GSH-Px水平,促进细胞存活,抑制细胞凋亡。miR-202-5p可靶向负性调控SOX6的表达。上调SOX6可部分逆转miR-202-5p对缺氧复氧处理心肌细胞的保护作用。结论:miR-202-5p通过靶向下调SOX6减轻缺氧复氧诱导的心肌细胞氧化应激,促进细胞存活,抑制细胞凋亡,发挥保护作用。

Objective:To investigate the effect of miR-202-5 p targeting SOX6 on oxidative stress induced by hypoxia-reoxygenation in cardiomyocytes.Methods:Rat embryonic cardiomyocytes H9 C2 were cultured in vitro to construct a model of hypoxia/reoxygenation injury in cardiomyocytes.H9 C2 cells were randomly divided into blank group, hypoxia-reoxygenation group, hypoxia-reoxygenation+miR-con group, hypoxia-reoxygenation+miR-202-5 p group, hypoxia-reoxygenation+miR-202-5 p+pcDNA group, hypoxia reoxygenation+miR-202-5 p+pcDNA-SOX6 group.The expression levels of miR-202-5 p and SOX6 in cardiomyocytes were detected by qRT-PCR and Western blot.The CCK-8 method was used to detect the survival rate of cardiomyocytes.Flow cytometry was used to detect cardiomyocyte apoptosis.Reactive oxygen species(ROS) levels, malondialdehyde(MDA),and glutathione peroxidase(GSH-Px) levels were measured by the kit.The dual luciferase reporter gene assay and Western blot were to verify the targeted and regulatory relationship between miR-202-5 p and SOX6.Results:Hypoxia-reoxygenation significantly inhibited the expression of miR-202-5 p, promoted the expression of SOX6,increased the levels of ROS and MDA,decreased the level of GSH-Px, inhibited cell survival and promoted apoptosis.Up-regulation of miR-202-5 p significantly reduced ROS and MDA levels, increased GSH-Px levels, promoted cell survival, and inhibited apoptosis.miR-202-5 p could target and negatively regulate the expression of SOX6.Up-regulation of SOX6 partially reversed the protective effect of miR-202-5 p on cardiomyocytes treated with hypoxia-reoxygenation.Conclusion:miR-202-5 p reduces the oxidative stress induced by hypoxia-reoxygenation, promotes cell survival, inhibits cell apoptosis and plays a protective role by down-regulating SOX6.