miR-486-5p在氧化应激引起人骨髓间充质干细胞凋亡中的作用

目的:观察microRNA-486-5p(miR-486-5p)在氧化应激引起人骨髓间充质干细胞(h MSCs)凋亡中的作用并探讨其作用机制。方法:h MSCs经培养鉴定后分为5组:空白对照组、H2O2组、miR-486-5p模拟物+H2O2组、抑制物(αnti-miR)+H2O2组及相应的阴性对照(scrambled control)+H2O2组。荧光定量PCR(real-time PCR)检测氧化应激诱导h MSCs凋亡过程中miR-486-5p的表达变化。用脂质体分别转染miR-486-5p的模拟物、抑制物及阴性对照到h MSCs。应用MTT、Hoechst标记和流式细胞术的方法检测miR-486-5p对氧化应激介导细胞活性下降及凋亡效应的影响,Western blotting检测凋亡相关蛋白、Akt与其磷酸化水平,采用试剂盒测定caspase-3活性。结果:H2O2诱导h MSCs凋亡过程中miR-486-5p的表达较对照组显著下降(P0.05)。与阴性对照组相比,在h MSCs中过表达miR-486-5p,能使细胞在氧化应激情况下活性显著下降,凋亡发生率增高,蛋白Bcl-2/Bax比值、caspase-3酶原含量及Akt磷酸化水平降低,caspase-3活性增强;而使用抑制物阻遏miR-486-5p的作用后,细胞在氧化应激条件下活性增加,凋亡发生率降低,蛋白Bcl-2/Bax比值及Akt磷酸化水平升高,caspase-3活性下降。结论:过表达miR-486-5p促进氧化应激引起的h MSCs凋亡,阻遏miR-486-5p的作用抑制氧化应激条件下的h MSCs凋亡,其中作用机制可能与调控Akt通路有关。

Role of miR-486-5p in apoptosis of human bone marrow mesenchymal stem cells induced by hydrogen peroxide

AIM: To investigate the role of microRNA-486-5p (miR-486-5p) in the apoptosis of human bone marrow mesenchymal stem cells (hMSCs) induced by hydrogen peroxide (H₂O₂). METHODS: The hMSCs were cultured in vitro and exposed to serum-free medium and H₂O₂ (10 mmol/L). The changes of miR-486-5p expression in oxidative stress-related apoptosis of hMSCs were measured by real-time PCR. The hMSCs were transfected with miR-486-5p mimic or inhibitor at concentration of 30 nmol/L by Lipofectamine RNAiMAX. The effect of miR-486-5p on H₂O₂-induced decrease in cell viability was evaluated by MTT assay. Hoechst 33342 staining and flow cytometry were applied to determine the role of miR-486-5p in the apoptosis of hMSCs. The protein expression was evaluated by Western blotting. Caspase-3 activity was determined using a caspase-3 activity kit. RESULTS: Compared with control group, the expression of miR-486-5p significantly decreased after treated with H₂O₂ (P<0.05). In addition, over-expression of miR-486-5p in the hMSCs reduced the cell viability, accelerated apoptosis, down-regulated Bcl-2/Bax ratio, caspase-3 enzyme precursor content and phosphorylation of Akt, and activated caspase-3 activity. Conversely, down-regulation of miR-486-5p significantly inhibited H₂O₂-induced cell apoptosis and the caspase-3 activity, increased cell viability and up-regulated Bcl-2/Bax ratio and phosphorylation level of Akt. CONCLUSION: Over-expression of miR-486-5p promotes H₂O₂-induced hMSCs apoptosis, and repression of miR-486-5p protects hMSCs from H₂O₂-induced cellular apoptosis, which may be mediated by regulating Akt signaling pathway.