下调microRNA-181b在小鼠缺血性脑损伤中的神经保护作用

Down-regulation of microRNA-181b has protective effect on cerebral ischemic injury of mice

目的:探讨microRNA-181b(miR-181b)在缺血性小鼠脑损伤病理过程中的神经保护作用。方法:应用N2A细胞氧-糖剥夺(OGD)模型模拟神经细胞缺血损伤;应用小鼠大脑中动脉阻塞(MCAO)模型模拟缺血性脑损伤(成功率约60%),原位细胞凋亡检测试剂盒检测N2A细胞损伤程度,蛋白印迹检测miR-181b靶蛋白自噬蛋白5(Atg5)及caspase-9的变化情况,萤光素酶报告基因技术检测miR-181b对Atg5 mRNA的直接调控作用。结果:在OGD致N2A细胞缺血损伤过程中,通过上调或抑制miR-181b的表达水平可以显著影响N2A细胞的凋亡(P<0.05);miR-181b表达水平的改变可显著影响Atg5蛋白表达水平(P<0.05);共转染miR-181b前体或miR-181b抑制剂可显著抑制或增高含有Atg5 mRNA 3’-UTR的萤光素酶报告基因的活性(P<0.05);MCAO后活化caspase-9的表达显著升高,而miR-181b拮抗剂可使MCAO后剪切的caspase-9表达明显减少(P<0.05)。结论:下调miR-181b可能通过调节Atg5的蛋白表达在缺血性小鼠脑损伤中发挥神经保护作用。

AIM： To explore the role of microRNA-181b（ miR-181b） in ischemic injury and autophagy protein 5（ Atg5） levels of mice. METHODS： Oxygen- glucose depletion（ OGD） model in N2 A cells to mimic ischemic injury in vitro was established. A middle cerebral artery occlusion（ MCAO） model to mimic ischemic injury in vivo was also induced in mice. The N2 A cell apoptosis after OGD was assessed by in situ cell death detection kit. The Atg5 and caspase-9 expressions were determined by Western blotting. Luciferase reporter assay was performed to identify the direct binding of miR-181 b with 3＇-UTR of Atg5 mRNA. RESULTS： The alteration of miR-181 b expression level by transfection with premiR-181 b or anti-miR-181 b significantly affected N2 A cell apoptosis（ P〈0. 05）. Accordingly,the changes of miR-181 b levels significantly altered the protein level of Atg5（ P〈0. 05）. Co-transfection of the luciferase reporters with pre-miR-181 b or anti-miR-181 b resulted in the inhibition or enhancement of the luciferase activities of luciferase expressing plasmid containing 3＇-UTR of Atg5 mRNA（ P〈0. 05）. In addition,the miR-181 b antagonist significantly reduced the cleaved caspase-9 levels in cerebral ischemic cortex of the mice after MCAO（ P〈0. 05）. CONCLUSION： Down-regulation of miR-181 b plays an important role in ischemic injury of mice through regulating Atg5 protein level.