过表达微小RNA-130a-3P对LPS诱导心肌细胞损伤的干预作用及其机制

Overexpression of miRNA-130a-3p alleviates LPS-induced cardiomyocyte injury by regulating autophagy and apoptosis

目的:探讨miRNA-130a-3p对脂多糖(LPS)诱导的心肌细胞自噬与凋亡的影响及分子机制。方法:H9C2心肌细胞随机分为5组,即正常对照组,LPS模型组,miRNA阴性对照组(miRNA-negative control组),miRNA-130a-3p mimics组(过表达miRNA-130a-3p),miRNA-130a-3p mimics+LY294002组(过表达miRNA-130a-3p+PI3K抑制)。LPS模型组即终浓度为10μg/ml的LPS诱导24 h,miRNA阴性对照组与miRNA-130a-3p mimics组是利用lipo3000将阴性对照miRNA及miRNA-130a-3p mimics转染至H9C2细胞,培养24 h后,再将LPS加入培养基中培养24 h。miRNA-130a-3p mimics+LY294002组是利用lipo3000将miRNA-130a-3p mimics转染至H9C2细胞,同时在培养基中加入10μmol/L(终浓度)的LY294002,培养24 h后,再将浓度为10μg/ml的LPS加入培养基中培养24 h。所有实验均重复5次以上。利用RT-qPCR检测细胞中miRNA-130a-3p mRNA的表达水平,利用CCK-8实验检测细胞活性,利用ELISA实验检测细胞培养液中肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),白细胞介素-1β(IL-1β)的含量,利用比色法检测细胞培养液中超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)的含量;利用Western blot检测细胞中p-PI3K蛋白,p-AKT蛋白,Bax蛋白,Bcl-2蛋白,cleaved-caspase-3蛋白,LC3蛋白,p62蛋白的表达水平。结果:结果显示,与正常组相比较,LPS模型细胞中miRNA-130a-3p mRNA水平,p-PI3K蛋白与p-AKT蛋白的水平显著低于正常对照组(P<0.01);与LPS组相比较,miRNA-130a-3p mimics组细胞中p-PI3K,p-AKT蛋白的表达显著升高(P<0.01,P<0.05);与正常对照组相比较,LPS组细胞活性显著降低,细胞培养液中TNF-α,IL-6,IL-1β及LDH的含量显著升高(P<0.01),SOD的含量显著降低(P<0.01),细胞中Bax蛋白,cleaved caspase-3蛋白,p62蛋白的表达显著升高(P<0.01),Bcl-2蛋白的表达和LC3II/I的比率显著降低(P<0.01);与LPS组相比较,miRNA-130a-3p mimics可提高细胞活性,降低细胞培养液中TNF-α,IL-6,IL-1β及LDH的含量(P<0.01,P<0.05),提高SOD的含量(P<0.05),降低细胞中Bax蛋白,cleaved caspase-3蛋白,p62蛋白的表达(P<0.01),促进Bcl-2蛋白的表达(P<0.01),提高LC3II/I的比率(P<0.05);与miRNA-130a-3p mimics组相比较,miRNA-130a-3p mimics+LY294002组,可部分逆转miRNA-130a-3p mimics对细胞的作用。结论:过表达miRNA-130a-3p可部分通过激活PI3K/AKT信号通路促进细胞的自噬与抑制细胞凋亡,减轻LPS诱导的心肌细胞损伤。

Objective:To investigate the effects of miRNA-130 a-3 p on autophagy and apoptosis induced by LPS in myocardial cells and its molecular mechanisms.Methods:H9 C2 cells were divided into five groups:normal control group,LPS model group,miRNA negative control group miRNA-130 a-3 p mimics group(overexpression of miRNA-130 a-3 p)and miRNA-130 a-3 p mimics+LY294002 group(overexpression of miRNA-130 a-3 p+PI3 K inhibitor).The LPS model group was induced by LPS at a final concentration of 10μg/ml for 24 h.In the miRNA negative control group and miRNA-130 a-3 p mimics group,negative contro miRNA or miRNA-130 a-3 p mimics were transfected into H9 C2 cells by lipo3000.After 24 h of culture,LPS was added into the medium for 24 hours.In the miRNA-130 A-3 P mimics+LY294002 group,miRNA-130 A-3 P mimics was transfected into H9 C2 cells by using lipo3000,and LY294002 at a final concentration of 10μmol/L was added to the culture medium for 24 h,followed by LPS at a concentration of 10μg/ml for 24 h.The expression of miRNA-130 a-3 p mRNA in cells was detected by RT-qPCR.The CCK-8 assay was used to detect the cell viability.The contents of TNF-α,IL-6 and IL-1βwere detected by ELISA assay.The contents of SOD and LDH in cell culture medium were detected by colorimetry.Western blot was used to detect the protein expressions of p-PI3 K,p-AKT,Bax,Bcl-2,cleaved-caspase-3,LC3 and p62.Results:The results showed that the levels of miRNA-130 a-3 p mRNA,p-PI3 K protein and p-AKT protein in LPS model cells were significantly lower than those in normal control group(P<0.01),and the expressions of p-PI3 K,p-AKT protein in miRNA-130 a-3 p mimics group were increased significantly compared with LPS group(P<0.01,P<0.05).Compared with normal control group,the cell viability was decreased significantly and the contents of TNF-α,IL-6,IL-1βand LDH were increased significantly(P<0.01),the contents of SOD was decreased significantly in LPS group(P<0.01).The protein expression levels of Bax,cleaved-caspase-3 and p62 were increased significantly,while the expression level of Bcl-2 and LC3 II/I ratio were decreased significantly in LPS group(P<0.01).miRNA-130 a-3 p mimics could increase the cell viability,decrease the contents of TNF-α,IL-6,IL-1βand LDH(P<0.01,P<0.05),increase the contents of SOD(P<0.05),decrease the expressions of Bax,cleaved caspase-3,p62(P<0.01),promote the expression of Bcl-2(P<0.01)and increase the ratio of LC3 II/I(P<0.05).Compared with miRNA-130 a-3 p mimics group,LY294002 reversed the effects of miRNA-130 a-3 p mimics on cells.Conclusion:Overexpression miRNA-130 a-3 p could partly promote autophagy and inhibit cell apoptosis by activating PI3 K/AKT signaling pathway to alleviate LPS-induced myocardial injury.