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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) overexpression on Nestin expression in mouse brain cortex neural stem cells (NSCs). Methods NSCs of fetal rat cerebral cortex were primary cultured by neurosphere formation test and transfected with miR-34a mimics (miR-34a mimics) and its negative control (mmu -miR-34a control). The liposome control group (transfected with liposome only) and blank control group were also established. After 48 h of transfection, real-time RT-PCR was used to detect the expression of miR-34a in each group And Nestin mRNA transcription level, Western blot detection of Nestin protein expression levels. Results miR-34a mimics upregulated the expression of miR-34a in NSCs (P <0.01). The overexpression of miR-34a down-regulated the expression of Nestin mRNA and protein All <0.01). Conclusion Exogenous overexpression of miR-34a down-regulates Nestin mRNA expression and protein expression in NSCs, providing a new theoretical basis for the regulation of proliferation and differentiation of NSCs.