FoxO4靶向siRNA通过减少自噬抑制小鼠神经干细胞向神经元分化

FoxO4-targeted siRNA inhibits neuronal differentiation of mouse neural stem cells by decreasing autophagy

目的:探讨FoxO4是否通过调控自噬影响小鼠神经干细胞(NSCs)的分化能力,寻找调控NSCs分化潜能的有效途径.方法:利用新生BALB/c小鼠提取海马齿状回(DG)NSCs,通过real time RT-PCR和Western Blot实验检测NSCs在分化前后自噬相关基因以及转录因子Forkhead Box O家族(FoxO3、FoxO4和FoxO6)的表达水平;运用基因沉默技术,将电转染后的NSCs分为NC-siRNA组和FoxO4-siRNA组,检测两组NSCs FoxO4 mRNA及蛋白的表达水平,从而验证已转入siRNA序列的目的基因沉默效率;通过real time RT-PCR和Western Blot实验检测NC-siRNA组和FoxO4-siRNA组NSCs自噬相关基因的表达;进一步将两组NSCs分化培养后通过real time RT-PCR实验以及免疫荧光染色检测Tuj1的表达.结果:体外分离培养P0小鼠海马DG区NSCs分化后自噬相关基因ATG3、ATG5、ATG7和Beclin-1 mRNA以及LC3-Ⅱ/Ⅰ蛋白表达水平被观察到均升高(P<0.01),同时还观察到NSCs分化后FoxO3、FoxO4、FoxO6 mRNA的表达水平均增高(P<0.01),其中转录因子FoxO4的mRNA和蛋白表达增高最为显著(P<0.01).通过运用靶向FoxO4的siRNA转染NSCs后,发现NSCs自噬相关基因ATG3、ATG5、ATG7和Beclin-1 mRNA以及LC3-Ⅱ/Ⅰ蛋白表达水平均降低(P<0.05),且NSCs中Tuj1表达受到显著抑制(P<0.05).结论:FoxO4基因沉默降低了小鼠海马NSCs中自噬水平,并抑制其向神经元分化.

Objective:To explore whether forkhead box protein O4(FoxO4) affects the differentiation ability of mouse neural stem cells(NSCs) by regulation of autophagy, and to find an effective way to regulate the differentiation potential of NSCs. Methods: The expression of autophagy-related genes and forkhead box O transcription factor family(FoxO3, FoxO4, and FoxO6) in undifferentiation and differentiation of NSCs cultured in the hippocampal dentate gyrus(DG) area of newborn BALB/c mice in vitro, were detected by real time RT-PCR and Western Blot experiments. Aiming at determining the silencing efficiency of siRNA sequence that have been turned into target genes, we divided NSCs into NC-siRNA group and FoxO4-siRNA group after NSCs electrotransfection and respectively detected the expression of FoxO4 mRNA and protein in two groups by gene silencing technique. The expression of autophagy-related genes in NSCs in the NC-siRNA group and the FoxO4-siRNA group were detected by real time RT-PCR and Western Blot experiments. Additionally, the expression of Tuj1 in NSCs of the two groups was further detected by real time RT-PCR and immunofluorescence staining after differentiation culture. Results: After NSCs differentiation in P0 mice DG region in vitro, that ATG3, ATG5, ATG7, Beclin-1 mRNA and LC3-Ⅱ/Ⅰ protein such as autophagy-related genes expression levels were observed to increase(P<0.01), in the meantime, that the expression levels of FoxO3, FoxO4, and FoxO6 mRNA were also found to increase(P<0.01), especially the expression levels of transcription factor FoxO4 mRNA and protein remarkably increased(P<0.01). By transfecting NSCs with siRNA targeting FoxO4, we found that the expression of NSCs autophagy-related genes decreased(P<0.05), such as ATG3, ATG5, ATG7,Beclin-1 mRNA and LC3-Ⅱ/Ⅰ protein, and the expression of Tuj1 in NSCs significantly decreased(P<0.05). Conclusion:FoxO4 gene silencing reduced the level of autophagy and inhibited neuronal differentiation in mouse hippocampal NSCs.