miR-124 facilitates the proliferation and differentiation of neural stem cells by inhibiting the Notch pathway

doi:10.3969/j.issn.2095-4344.0669

Abstract

Abstract:

BACKGROUND: How to quickly and efficiently obtain enough pure neurons in vitro is a huge challenge and problem faced by the scientists in the cell-transplantation field.
OBJECTIVE: To explore whether miR-124 can influence the proliferation and differentiation of neural stem cells by regulating the Notch pathway.
METHODS: The fetal rat neural stem cells were isolated and identified by immunofluorescence. RT-qPCR was used to detect the expression of miR-124 after 1, 2, 4, and 7 days of neural stem cell differentiation. After overexpression and interference with miR-124 via transient transfection, the expression of ki-67 protein was detected by MTT assay and immunoblotting. RT-qPCR and immunoblotting were used to detect β-tubulin III expression for determination of miR-124 effects on cell differentiation. RT-qPCR was used to detect the mRNA expression of Notch pathway-related proteins HES1, HEY2 and CCND1.
RESULTS AND CONCLUSION: During the differentiation of neural stem cells into neurons, the expression of miR-124 was significantly upregulated. Moreover, miR-124 overexpression promoted the proliferation and differentiation of NSCs, and suppressed the expression of proteins in the Notch pathway. Silencing the miR-124 expression, however, achieved the opposite results. The above results suggest that miR-124 may promote the proliferation and differentiation of neural stem cells by inhibiting the Notch pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: MicroRNAs, Neural Stem Cells, Receptors, Notch, Cell Proliferation, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

He Pin, Guo Fu-qiang. miR-124 facilitates the proliferation and differentiation of neural stem cells by inhibiting the Notch pathway[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5344-5349.

Figures/Tables 2

2.1 大鼠神经干细胞形态及鉴定结果分离的大鼠神经干细胞保持自我增殖和未分化状态，主要以神经球的形式贴壁生长，细胞排列紧密，克隆边缘清晰(图1A)。Nestin是神经干细胞的重要检测指标，细胞传代后24 h，利用免疫荧光法对细胞进行染色鉴定，约有90%以上神经干细胞为Nestin阳性，且与细胞核共定位(图1B-D)，说明分离的细胞为神经干细胞。 2.2 miR-124在大鼠神经干细胞中的表达神经干细胞向神经细胞诱导分化1，2，4，7 d后，miR-124的表达水平随着时间推移显著升高(图2A)；然后通过瞬时转染miR-124 mimic过表达miR-124以及转染miR-124 inhibitor干扰miR-124表达，在转染48 h之后，过表达组和干扰组的miR-124水平与空载对照组比较差异有显著性意义(P < 0.01，图2B，C)，提示转染效果确切。 2.3 miR-124对神经干细胞增殖的影响神经干细胞分别转染miR-124 mimic和miR-124 inhibitor 24，48，72 h后，采用MTT法检测各组细胞的体外增殖情况，在miR-124 mimic转染24 h后，miR-124过表达组的细胞增殖速度显著高于无意义链对照组(图3A)；在miR-124 inhibitor转染48 h后，miR-124干扰组的细胞增殖速度显著低于无意义链对照组(图3B)。为进一步验证上述实验结果，神经干细胞分别转染miR-124 mimic和miR-124 inhibitor 48 h后，又采用免疫印迹检测法检测各组细胞的细胞增殖标记物ki-67的表达水平(图3C和3D)，与MTT法检测结果类似。以上结果提示，miR-124表达可以显著促进大鼠神经干细胞在体外的增殖。 2.4 miR-124对神经干细胞分化的影响神经干细胞分别转染miR-124 mimic和miR-124 inhibitor 48 h后，转染miR-124 mimic组神经元特异标记分子β-tubulin Ⅲ的mRNA(图4A)和蛋白(图4C)表达水平较对照组均显著升高。转染miR-124 inhibitor组β-tubulin Ⅲ的mRNA(图4B)和蛋白(图4D)表达水平较对照组均显著下降，提示miR-124可促进神经干细胞向神经元分化。 2.5 miR-124对神经干细胞Notch通路的影响研究发现，Notch信号通路能够通过调节靶基因的表达，进而影响细胞的增殖、存活、凋亡等生理过程。在转染miR-124 mimic和miR-124 inhibitor 48 h后，通过RT-qPCR检测Notch信号通路下游靶基因HES1，HEY2和CCND1 mRNA表达。结果显示，转染miR-124 mimic后，HES1(图5A)，HEY2(图5B)和CCND1(图5C)的mRNA表达水平较对照组均显著降低。转染miR-124 inhibitor后，HES1(图5D)，HEY2(图5E)和CCND1(图5F)的mRNA表达水平较对照组均显著升高。以上结果提示，miR-124在体外促进神经干细胞的增殖和分化的作用可能是通过抑制Notch信号通路来实现的。"

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