ABSTRACT

microRNAs have been proved to function in some processes of differentiation and the effect is favorable. At present, the differentiation of stem cells is not so ideal because of the high expenses and inaccessibility. Therefore, we explored the possibility that microRNA-221 (miR-221) affects differentiation from stem cells from human deciduous tooth (SHEDs) to neurons through Wnt/β-catenin pathway via binding to CHD8. After collection of SHEDs, differentiation from SHEDs to neurons was conducted by neurotrophic factor induction method in vitro, followed by gain- and loss-of-function experiments. Expression of neuron-related genes in SHEDs was examined by immunohistochemistry. The relationship between CHD8 and miR-221 was detected by dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to determine miR-221 expression, and the mRNA and protein expression of CHD8, Wnt/β-catenin pathway- and neuron-related genes. Cell viability, and cell cycle and apoptosis were investigated by MTT assay and flow cytometry respectively. Dual luciferase reporter assay displayed that miR-221 targeted CHD8 and then affected the differentiation progression. Results of RT-qPCR and Western blot analysis showed that expression of Wnt/β-catenin pathway-related genes increased significantly, CHD8 expression decreased in neuron-induced SHEDs after miR-221 overexpression or CHD8 silencing. In response to miR-221 overexpression and CHD8 silencing, cell viability and cell cycle entry were increased, and apoptosis was reduced. Moreover, overexpression of miR-221 or silencing of CHD8 elevated the expression of neuron-related genes in neuron-induced SHEDs. Taken together, upregulation of miR-221 promotes differentiation from SHEDs to neuron cells through activation of Wnt/β-catenin pathway by binding to CHD8.

 抽象的

microRNA已被证明在某些分化过程中发挥作用，且效果良好。目前，干细胞的分化由于费用高、获取困难等原因还不太理想。因此，我们探讨了microRNA-221（miR-221）通过与CHD8结合，通过Wnt/β-catenin途径影响人乳牙（SHED）干细胞向神经元分化的可能性。收集SHED后，通过神经营养因子诱导法在体外进行SHED向神经元的分化，然后进行功能获得和丧失实验。通过免疫组织化学检查 SHED 中神经元相关基因的表达。通过双荧光素酶报告基因检测检测CHD8和miR-221之间的关系。 RT-qPCR 和 Western blot 分析用于测定 miR-221 表达以及 CHD8、Wnt/β-catenin 通路和神经元相关基因的 mRNA 和蛋白表达。分别通过MTT法和流式细胞术研究细胞活力、细胞周期和凋亡。双荧光素酶报告基因检测显示 miR-221 靶向 CHD8，然后影响分化进程。 RT-qPCR和Western blot分析结果显示，在miR-221过表达或CHD8沉默后，神经元诱导的SHED中Wnt/β-catenin通路相关基因的表达显着增加，CHD8表达降低。响应 miR-221 过表达和 CHD8 沉默，细胞活力和细胞周期进入增加，细胞凋亡减少。此外，miR-221的过度表达或CHD8的沉默会升高神经元诱导的SHED中神经元相关基因的表达。 综上所述，miR-221 的上调通过与 CHD8 结合激活 Wnt/β-catenin 通路，促进 SHED 分化为神经元细胞。