微RNA-24对过氧化氢诱导的人脐静脉血管内皮细胞凋亡的影响及作用机制

Effects and mechanism of microRNA-24 on hydrogen peroxide-induced apoptosis of human umbilical vein endothelial cells

目的探讨微RNA-24(miR-24)对H2O2诱导的人脐静脉血管内皮细胞(HUVEC)存活、迁移和凋亡的影响及作用机制。方法通过转染miR-24高表达、miR-24抑制物(anti-miR-24)及其阴性对照慢病毒质粒(miR-24 NC)构建稳转细胞,3种细胞用H2O2500μmol·L^-1处理12 h。采用四甲基偶氮唑盐(MTT)法检测细胞存活率,划痕实验检测细胞迁移率,Hoechst33258染色及流式细胞术检测细胞凋亡,实时荧光定量聚合酶链反应(qRT-PCR)检测miR-24,Bax,Bcl-2和胱天蛋白酶3 mRNA表达水平,Western印迹法和免疫细胞化学法检测Bax、Bcl-2和胱天蛋白酶3蛋白表达水平。结果与miR-24 NC组比,miR-24高表达组miR-24表达升高(P<0.05),而anti-miR-24组降低(P<0.05)。与正常对照组比,H2O2组细胞凋亡率增加(P<0.05)、细胞存活和迁移率降低(P<0.05),表明氧化损伤模型构建成功。与H2O2+miR-24 NC组比,H2O2+miR-24高表达组上述指标较H2O2+miR-24 NC组升高(P<0.05),促凋亡蛋白Bax和胱天蛋白酶3 mRNA与蛋白表达量增加,抑凋亡蛋白Bcl-2 mRNA和蛋白表达量降低(P<0.05),而H2O2+anti-miR-24组可降低细胞凋亡率、增加细胞存活和迁移率,降低Bax和胱天蛋白酶3 mRNA与蛋白表达水平,增加Bcl-2 mRNA和蛋白表达水平(P<0.05)。结论氧化应激状态下,miR-24可通过上调Bax、胱天蛋白3表达和下调Bcl-2蛋白表达,促进HUVEC凋亡和抑制其存活和迁移能力。

OBJECTIVE To investigate the effects and mechanism of microRNA-24(miR-24)on viability,migration and apoptosis of human umbilical vein endothelial cells(HUVECs)induced by hydrogen peroxide(H2O2).METHODS Stable transfectant cell lines were transfected with miR-24 high expression,miR-24 inhibitor(anti-miR-24)and negative control lentiviral plasmids(miR-24 NC),respectively,and the cells were treated with H2O2500μmol·L^-1 for 12 h.The viability of cells was evaluated by MTT assay,while the ability of cell migration was evaluated by the wound healing.Cell apoptosis was determined by Hoechst33258 stain and flow cytometry.The expression levels of miR-24,Bax,Bcl-2 and caspase 3 mRNA were detected by quantitative real-time polymerase chain reaction(qRT-PCR),whereas the protein expressions of Bax,Bcl-2 and caspase 3 were detected by Western blotting and Immunocytochemistry respectively.RESULTS Compared with miR-24 NC group,the expression of miR-24 in the miR-24 high-expression group was markedly increased(P<0.05),but was decreased in the anti-miR-24 group(P<0.05).Compared with normal control group,the apoptosis rate of H2O2 group was increased(P<0.05),but the viability and migration of HUVECs were decreased(P<0.05),suggesting that an oxidative injury model was successfully constructed.The above indexes in the H2O2+miR-24 high-expression group were higher than in the H2O2+miR-24 NC group(P<0.05).Additionally,the mRNA and protein expressions of Bax and caspase 3 were obviously increased,but Bcl-2 expression was decreased(P<0.05).The apoptosis rate of the H2O2+anti-miR-24 group was decreased,but cell viability and the ability of migration increased.The mRNA and protein expressions of Bax and caspase 3 decreased,while the Bcl-2 expression increased(P<0.05).CONCLUSION miR-24 can promote HUVECs apoptosis and inhibit cell viability and migration under oxidative damage by up-regulating the expressions of Bax,caspase 3 and down-regulating the expression of Bcl-2 protein.