微小核糖核酸-125b-5p抑制Caspase 2蛋白酶活性缓解脂多糖诱导的心肌细胞凋亡和氧化应激的研究

miR-125b-5p inhibits Caspase 2 protease activity to relieve Lipopolysaccharide-induced cardiomyocyte apoptosis and oxidative stress

目的:MicroRNAs是一种转录后调控靶基因的非编码小RNA,被认为是心脏功能和疾病的重要调节剂,心脏损伤伴随着MicroRNAs表达的动态变化。本研究以脂多糖(LPS)诱导损伤制备H9c2心肌细胞氧化应激性损伤模型,探究miR-125b-5p对Caspase 2蛋白酶活性和LPS诱导的心肌细胞凋亡和氧化应激的影响。方法:采用LPS诱导损伤制备H9c2心肌细胞氧化应激性损伤模型,将miR-125b-5p转染于LPS诱导的H9c2心肌细胞中。RT-PCR检测miR-125b-5p和Caspase 2的表达水平;荧光素酶报告实验确定miR-125b-5p和Caspase 2的靶向关系;流式细胞术检测细胞凋亡;免疫印迹检测Caspase-2,Caspase-3,Caspase-9,Survivin,Bax/Bcl-2的蛋白表达;试剂盒检测细胞培养液中乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)活性以及丙二醛(MDA)含量。结果:用LPS诱导处理能显著降低miR-125b-5p的表达水平(P<0.05),促进Caspase 2表达(P<0.05)。荧光素酶报告实验表明Caspase 2上存在miR-125b-5p的结合位点,miR-125b-5p能靶向抑制Caspase 2活性(P<0.05)。miR-125b-5p可显著抑制Caspase 2蛋白表达水平(P<0.05)。miR-125b-5p能明显促进H9c2心肌细胞增殖(P<0.05)。miR-125b-5p能显著抑制H9c2心肌细胞凋亡(P<0.05)。同时,miR-125b-5p还能显著抑制裂解的caspase-3、caspase-9和Bax/Bcl-2蛋白水平(P<0.05),促进Survivin的蛋白表达(P<0.05)。miR-125b-5p可明显降低培养液中LDH活性和细胞内MDA、GSH含量、增加胞内SOD活性(P<0.05)。结论:miR-125b-5p通过抑制Caspase 2蛋白酶活性,促进心肌细胞增殖,降低心肌细胞凋亡率,缓解了LPS诱导的心肌细胞凋亡和氧化应激,表明miR-125b-5p对LPS造成的心肌细胞损伤具有保护作用。

Objective:MicroRNAs are small non-coding RNA that regulate target genes after transcription and are considered as important regulators of heart function and disease.Cardiac injury is accompanied by dynamic changes in MicroRNAs expression.In this study,the oxidative stress injury model of H9c2 cardio-myocytes was prepared by LPS-induced injury to investigate the effects of miR-125b-5p on Caspase 2 protease activity and LPS-induced apoptosis and oxidative stress of cardiomyocytes.Methods:The oxidative stress injury model of H9c2 cardiomyocytes was established using LPS-induced injury,and miR-125b-5p was transfected into LPS-induced H9c2 cardiomyocytes.The expression levels of miR-125b-5p and Caspase 2 were detected by RT-PCR.Luciferase reporting assay determined the targeting relationship between miR-125b-5p and Caspase 2.Apoptosis was detected by flow cytometry.Western blotting was used to detect the expression of Caspase-2,Caspase-3,Caspase-9,Survivin and Bax/Bcl-2.The activity of LDH,S0D and MDA in cell culture were detected by the kit.Results:LPS induced treatment significantly reduced the expression level of miR-125b-5p(P<0.05)and promoted the expression of Caspase 2(P<0.05).Luciferase reporting experiments showed the presence of binding sites for miR-125b-5p on Caspase 2,which could inhibit Caspase 2 activity(P<0.05).miR-125b-5p significantly inhibited the expression of Caspase 2 protein(P<0.05).miR-125b-5p significantly promoted the proliferation of H9c2 cardiomyocytes(P<0.05).miR-125b-5p significantly inhibited apoptosis of H9c2 cardiomyocytes(P<0.05).At the same time,miR-125b-5p can significantly inhibit the protein levels of caspase-3,caspase-9 and Bax/bcl-2,and promote the protein expression of Survivin(P<0.05).miR-125b-5p can significantly reduce LDH activity and intracellular MDA and GSH content,and increase intracellular SOD activity(P<0.05).Conclusions:miR-125b-5p promotes cardiomyocyte proliferation,reduces cardiomyocyte apoptosis rate,and attenuates LPS-induced cardiomyocyte apoptosis and oxidative stress by inhibiting Caspase 2 protease activity,indicating that cardiomyocytes induced by LPS by miR-125b-5p Damage has a protective effect.