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

摘要

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 cul-tured in vitro and exposed to serum-free medium and H2O2(10 mmol/L).The changes of miR-486-5p expression in oxida-tive 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 H2 O2-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 ac-tivity 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 H2 O2-induced cell apoptosis and the caspase-3 activity, increased cell viability and up-regulated Bcl-2/Bax ratio and phos-phorylation level of Akt.CONCLUSION:Over-expression of miR-486-5p promotes H2 O2-induced hMSCs apoptosis, and repression of miR-486-5p protects hMSCs from H2 O2-induced cellular apoptosis, which may be mediated by regulating Akt signaling pathway.%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（hMSCs）凋亡中的作用并探讨其作用机制。方法：hMSCs经培养鉴定后分为5组：空白对照组、H2 O2组、miR-486-5p模拟物＋H2 O2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hMSCs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hMSCs。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hMSCs凋亡过程中miR-486-5p的表达较对照组显著下降（P＜0.05）。与阴性对照组相比，在hM-SCs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hMSCs凋亡，阻遏miR-486-5p的作用抑制氧化应激条件下的hMSCs凋亡，其中作用机制可能与调控Akt通路有关。