miR-200b-Notch信号通路对诱导性多能干细胞向神经干细胞分化的调控作用

Regulation of miR-200b-Notch Signaling Pathway on the Differentiation of Induced Pluripotent Stem Cells into Neural Stem Cells

目的探究miR-200b靶向Hes1调控Notch信号通路对iPS细胞向神经干细胞分化的影响。方法采用结合维甲酸和无血清培养基诱导法诱导iPS细胞向神经干细胞分化,分别在诱导分化的第0、4、8、15d采用实时荧光定量PCR检测miR-200b水平,Westernblot法检测Hes家族bHLH转录因子1(Hes1)、Notch1蛋白水平,应用生物信息学软件预测并通过荧光素酶报告实验验证miR-200b与Hes1之间的靶向结合作用。结果在iPS细胞向神经干细胞分化诱导分化的第0、4、8、15d,miR-200b水平逐渐升高,Hes1、Notch1蛋白水平逐渐降低,差异有统计学意义(P<0.05);生物信息学分析显示,Hes1为miR-200b的靶基因,过表达miR-200b可升高神经干细胞分化,上调Hes1水平调控Notch信号通路相关分子。结论miR-200b可促进iPS细胞向神经干细胞分化,其机制可能与其靶向上调Hes1表达,进而促进Notch信号通路的激活有关。

Objective To explore the effect of miR-200b targeting Hes1 to regulate the Notch signaling pathway on the differentiation of iPS cells into neural stem cells.Methods Combining retinoic acid and serum-free medium was used to induce iPS cells to differentiate into neural stem cells.Real-time fluorescence quantitative PCR was used to detect the level of miR-200b on the 0th,4th,8th,and 15th d of the induced differentiation,and the Hes family was detected by Western blot method.The bHLH transcription factor 1(Hes1)and Notch1 protein levels were predicted by bioinformatics software and the targeted binding effect between miR-200b and Hes1 was verified through the luciferase report experiment.Results On the 0th,4th,8th,and 15th d after the differentiation of iPS cells into neural stem cells,the level of miR-200b gradually increased,and the levels of Hes1 and Notch1 protein gradually decreased,the difference was statistically significant(P<0.05);Bioinformatics analysis showed that Hes1 is the target gene of miR-200b.Overexpression of miR-200b could increase the differentiation of neural stem cells and up-regulate Hes1 levels to regulate Notch signaling pathway related molecules.Conclusion miR-200b could promote the differentiation of iPS cells into neural stem cells,and its mechanism might be related to its targeted up-regulation of Hes1 expression,which in turn promoted the activation of Notch signaling pathway.