miR-34a过表达对小鼠脑皮质神经干细胞中巢蛋白表达的影响

Effect of miR-34a over-expression on Nestin expression in mouse cortex neutral stem cells

目的探讨miR-34a(microRNA-34a)过表达对小鼠脑皮质神经干细胞(Neutral stem cell,NSCs)标志物巢蛋白(Nestin)表达的影响。方法通过神经球(Neurosphere)形成试验原代培养ICR胎鼠脑皮质NSCs,在脂质体2000的介导下分别转染miR-34a模拟物(mmu-miR-34a mimics)及其阴性对照(mmu-miR-34a control),另设脂质体对照组(仅转染脂质体)及空白对照组,转染48 h后,采用real time RT-PCR检测各组细胞中miR-34a的表达水平及Nestin基因mRNA的转录水平,Western blot检测Nestin蛋白的表达水平。结果 miR-34a模拟物转染后,显著上调了miR-34a在NSCs中的表达水平(P<0.01);miR-34a的过表达显著下调了NSCs中Nestin基因mRNA转录及蛋白的表达水平(P均<0.01)。结论外源性过表达miR-34a可下调NSCs中Nestin基因mRNA的转录水平及蛋白的表达水平,为NSCs的增殖、分化调控的研究提供了新的理论依据。

Objective To investigate the effect of miR-34a（microRNA-34a） over-expression on Nestin expression in mouse cortex neutral stem cells（NSCs）.Methods Primary culture of fetal ICR mouse cortex NSCs were prepared by neurosphere-forming assay,and transfected with miR-34a mock（mmu-miR-34a mimics） and miR-34a control in mediation of liposome control respectively,using those transfected with liposome 2000 and those untransfected as control.The transcription levels of Nestin mRNA and miR-34a levels in various groups 48 h after transfection were determined by real time RT-PCR,while the expression level of Nestin by Western blot.Results Transfection with miR-34a significantly up-regulated the expression of miR-34a in NSCs（P 0.01）.However,the over-expression of miR-34a significantly down-regulated the transcription of Nestin mRNA and expression of Nestin in NSCs（both P 0.01）.Conclusion Over-expression of exogenous miR-34a down-regulated Nestin mRNA transcription and protein expression in NSCs,which provided a novel theoretical basis for study on regulation of proliferation and differen-tiation of NSCs.