miR-142-3p通过调控ELAVL1表达影响过氧化氢诱导的心肌细胞损伤的分子机制

目的探讨微小RNA-142-3p（miR-142-3p）对过氧化氢诱导的心肌细胞损伤的影响及其作用机制。 方法构建氧化应激损伤模型，以H9C2心肌细胞为研究对象，实验将心肌细胞转染后分为正常对照组、H₂O₂组、H₂O₂+miR-142-3p组、H₂O₂+miR阴性对照组、H₂O₂+?si-?ELAVL1组、H₂O₂+siRNA对照组、H₂O₂+miR-142-3p+pcDNA-ELAVL1组、H₂O₂+miR-?142-3p+pcDNA组。分别采用qRT-PCR与Western Blot检测细胞中miR-142-3p与ELAVL1表达；检测各组活性氧（ROS）生成水平；MTT检测细胞存活率，流式细胞术检测细胞凋亡。双荧光素酶报告实验验证miR-142-3p与ELAVL1的靶向作用。Western Blot检测细胞中Cleaved Caspase-3、STAT3、Caspase-3、p-STAT3蛋白表达。两组间比较采用两样本t检验；多组间比较采用单因素方差分析，两两比较采用LSD-t检验。 结果H₂O₂组心肌细胞中miR-142-?3p（0.26±0.06）、p-STAT3表达水平（0.36±0.04）、细胞存活率（61.73±6.48）﹪与正常对照组相比下降（P均< 0.01），而ROS水平（1?566.38±121.57）、细胞凋亡率（27.46±1.73）﹪、Cleaved Caspase-3（0.68±0.08）及ELAVL1表达水平（4.23±0.31）均升高（P均< 0.01）；双荧光素酶报告实验证实ELAVL1是miR-142-3p的靶基因；miR-142-3p过表达或沉默ELAVL1表达可明显促进心肌细胞存活、上调p-STAT3表达，而抑制细胞凋亡及Cleaved Caspase-3表达；ELAVL1过表达可逆转miR-142-3p对过氧化氢处理H9C2细胞的保护作用。 结论miR-142-?3p可通过抑制ELAVL1表达进而减轻过氧化氢诱导的心肌细胞损伤，其可能通过影响STAT3信号通路而保护心肌细胞。

Molecular mechanism of miR-142-3p in the injury induced by hydrogen peroxide by regulating ELAVL1

ObjectiveTo investigate the effect of microRNA-142-3p (miR-142-3p) on hydrogen peroxide-induced cardiomyocyte injury and its mechanism. MethodsThe oxidative stress injury model of H9C2 cardiomyocytes was established. H9C2 cardiomyocytes were randomly divided into normal control group, H₂O₂ group, H₂O₂+miR-142-3p group, H₂O₂+miR negative control group, H₂O₂+ si-ELAVL1 group, H₂O₂+ group. siRNA control group, H₂O₂+miR-142-3p+pcDNA-ELAVL1 group, H₂O₂+miR-142-3p+pcDNA group. qRT-PCR and Western Blot were used to detect the expression of miR-142-3p and ELAVL1 in H9C2 cardiomyocytes, respectively. Fluorescence probe method was used to detect the level of reactive oxygen species (ROS) production in H9C2 cardiomyocytes of each group. MTT assay and flow cytometry were used to detect H9C2 cardiomyocyte survival rate and apoptosis rate, respectively. The dual luciferase reporter assay validated the targeting of miR-142-3p and ELAVL1. Western Blot was used to detect the expression of Cleaved Caspase-3, STAT3, Caspase-3 and p-STAT3. ResultsThe expression of miR-142-?3p (0.26±0.06) , p-STAT3 expression (0.36±0.04) and cell viability (61.73±6.48) in the H₂O₂ group were significantly lower than those in the normal control group (P?< 0.01) . The ROS level (1566.38±121.57) , apoptosis rate (27.46±1.73) , Cleaved Caspase-3 (0.68±0.08) and ELAVL1 expression level (4.23±0.31) were significantly increased (P?< 0.01) . Dual luciferase reporter assay confirmed that ELAVL1 was a target gene of miR-142-3p. The miR-142-3p overexpression or silencing ELAVL1 could significantly promote the myocardial cell survival, up-regulate the p-STAT3 expression, while inhibit the cardiomyocyte apoptosis and expression of Cleaved Caspase-3.Overexpression of ELAVL1 reversed the protective effect of miR-142-3p on H9C2 cells treated with hydrogen peroxide. ConclusionmiR-142-3p may attenuate the hydrogen peroxide-induced cardiomyocyte injury by inhibiting the ELAVL1 expression, which may protect cardiomyocytes by affecting the STAT3 signaling pathway.