miR-221在H2O2诱导的大鼠心肌细胞损伤中的作用及机制研究

摘要

目的 探讨miR-221在过氧化氢(H2O2)诱导的大鼠心肌细胞(H9c2)损伤中的作用及机制研究.方法 MTT法检测不同浓度H2O2对H9c2细胞的损伤作用,RT-PCR法检测miR-221表达量.通过Lipofectamine 2000将miR-221 inhibitor及阴性对照转入H9c2心肌细胞,并将实验分为4组,正常对照组,模型对照组(H2O2组),阴性对照组(H2O2+阴性对照组),抑制组(H2O2+miR-221 inhibitor组).MTT法检测细胞活力,吖啶橙染色检测细胞凋亡情况,Western blot检测Bax,Bcl-2,10号染色体缺失的磷酸酶及张力蛋白同源的基因蛋白(PTEN)及p-蛋白激酶(AKT)表达.结果 0、25、50、100、200、400 μmol/L H2O2对H9c2细胞活力的抑制作用逐渐加强,其中200 μmol/L H2O2对细胞活力抑制程度适中,因此作为后续诱导剂量.与正常对照组比较,模型对照组及阴性对照组中miR-221表达量显著上调(P < 0.01),H9c2细胞活力降低(P < 0.01),细胞凋亡率显著提高(P < 0.01),Bax及PTEN表达量上调(P < 0.01),Bcl-2及p-AKT表达量下调(P < 0.01).与模型对照组及阴性对照组比较,抑制组中miR-221表达量显著下调(P < 0.01),H9c2细胞活力提高(P < 0.01),细胞凋亡率显著降低(P < 0.01),Bax及PTEN表达量下调(P < 0.01),Bcl-2及p-AKT表达量上调(P < 0.01).结论 miR-221低表达能显著抑制H2O2诱导的H9c2心肌细胞氧化应激损伤,与调控PTEN/AKT信号通路有关.%Objective To explore the role of miR-221 in the injury induced by hydrogen peroxide (H2O2) in rat myocardial cells (H9c2).Methods The viability of H9c2 cell induced by cell different concentrations of H2O2 was determined by MTT.The expression of miR-221 was detected by RT-PCR method.The miR-221 inhibitor and negative control were transferred into H9c2 cells by Lipofectamine 2000, then the cells were divided into normal control group, model control group (H2O2 group), negative control group (H2O2+ negative control group), inhibition group (H2O2+miR-221 inhibitor group).The cell viability was measured by MTT assay.Cell apoptosis was detected by acridine orange staining method.The expression of Bcl-2, Bax, phosphatase and tensin homolog deleted on chromosome ten (PTEN, p-protein kinase B (AKT) were assayed by Western Blot.Results 0,25,50,100,200,400 μmol/L H2O2 inhibited H9c2 cell activity gradually, of which 200 mol/L inhibition of cell viability moderate, so as a subsequent induction dose.Compared with normal control group, cell viability was decreased (P < 0.01), cell apoptotic rat was increased (P < 0.01), the expression of Bax and PTEN was upregulated (P < 0.01), the expression of Bcl-2 and p-AKT was downregulated (P < 0.01) in model control group and negative control group.Compared with model control group and negative control group, inhibition group proves the contrary.Conclusions Down-expression of miR-221 could significantly inhibit oxidative stress damage in H9c2 cells, which related to regulation of PTEN/AKT signal pathway.