MicroRNA-20 regulates directional differentiation of stem cells from human exfoliated deciduous teeth

doi:10.3969/j.issn.2095-4344.1761

Abstract

Abstract:

BACKGROUND: In recent years, microRNA-20 (miR-20) has been found to plays an important role in promoting the osteogenic differentiation of bone marrow mesenchymal stem cells and inflammatory periodontal ligament stem cells. However, whether it can regulate the differentiation of dental pulp stem cells into neurons has not been fully clarified.
OBJECTIVE: To investigate the mechanism by which miR-20 regulates the differentiation of stem cells from human exfoliated deciduous teeth towards neuronal cells through bone morphogenetic protein signaling pathway.
METHODS: Stem cells from human exfoliated deciduous teeth were separated and purified. Cell morphology was observed during cell induction and differentiation. Nestin, neuron-specific enolase, miR-20 and bone morphogenetic protein-2 expressions were detected. The stem cells from human exfoliated deciduous teeth were divided into five groups: negative control group (transfected with negative control sequence), miR-20 mimic group (transfected with miR-20 mimic for 48 hours), miR-20 inhibitor group (transfected with miR-20 inhibitor for 48 hours), pathway inhibition group (treated with bone morphogenetic protein pathway inhibitor for 10 days), and combined treatment group (transfected with miR-20 mimic for 24 hours and then treated with bone morphogenetic protein pathway inhibitor for 10 days). Subsequently, the expressions of bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, Nestin and neuron-specific enolase were detected using real-time PCR and western blot assay at 12 days of neuronal induction. Immunofluorescence was adopted to determine the expression of III β-tubulin. Golgi staining was performed to detect the number of dendritic spines in neurons.
RESULTS AND CONCLUSION: The stem cells from human exfoliated deciduous teeth were gradually differentiated into neuron-like cells. During the differentiation period, the expression of Nestin was increased and then decreased, but the expression levels of neuron-specific enolase, miR-20 and bone morphogenetic protein-2 were gradually increased. Compared with the negative control group, miR-20 overexpression up-regulated the levels of bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, III β-tubulin, Nestin and neuron-specific enolase as well as induced generation of neuronal dendritic spines. But inhibition of miR-20 or bone morphogenetic protein pathway down-regulated the protein expression of bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, III β-tubulin, Nestin and neuron-specific enolase and inhibited the generation of dendritic spines in neurons. At the same time, bone morphogenetic protein inhibitor reversed the inducement of stem cells from human exfoliated deciduous teeth to neurons caused by miR-20. Thus, miR-20 can induce the differentiation of stem cells from human exfoliated deciduous teeth towards neuron-like cells through activating the bone morphogenetic protein signaling pathway.

Key words: stem cells from human exfoliated deciduous teeth, neuronal cells, miR-20, neural differentiation, bone morphogenetic protein signaling pathway, III β-tubulin, neuronal cell dendritic spine, Natural Science Foundation of Hainan Province

CLC Number:

Deng Xuan, Yang Fang, Tang Xiqing, Zhao Yan. MicroRNA-20 regulates directional differentiation of stem cells from human exfoliated deciduous teeth[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3329-3335.

Figures/Tables 2

2.1 人乳牙牙髓干细胞向神经元诱导分化的形态变化及标志物表达人乳牙牙髓干细胞体积较小、轮廓清晰，大多数为梭形或多角形，少部分为卵圆形，高表达Vinmentin、CD44和CD29。在神经分化诱导培养基中培养第3天，细胞胞体收缩，变为神经球样且成团生长。在神经分化诱导培养基中培养第12天，突起成网状且相互连接，核大而圆，位于细胞中央，为神经元样细胞，见图1A。Nestin表达在分化期间先升高后降低，在诱导第6天表达最高，见图1B-D。NSE蛋白及mRNA表达水平随着培养时间延长而上调，见图1E-G。 2.2 人乳牙牙髓干细胞中miR-20和BMP2的表达水平在神经诱导分化第3天后miR-20表达逐步上调，BMP2 mRNA和蛋白表达随着培养时间延长逐渐上调且在培养第9天其表达趋于平缓，见图2。总体而言，二者表达呈时间依赖性上升。 2.3 miR-20通过骨形态发生蛋白信号通路诱导人乳牙牙髓干细胞分化为神经元 Nestin和NSE分别为神经干细胞和神经元的特异标志物。在诱导分化第12天，Real-time PCR检测显示：与阴性对照组相比，miR-20 mimic组和联合组miR-20表达显著上调，miR-20 inhibitor组miR-20表达显著下调，通路抑制剂组miR-20表达无明显变化。Western blot检测结果发现：与阴性对照组相比，miR-20 mimic组BMP2、BMPR2、Nestin和NSE蛋白表达显著上调，miR-20 inhibitor组和通路抑制剂组BMP2、BMPR2、Nestin和NSE蛋白表达显著下调。此外，联合组对上述因子的诱导效果能被BMP通路抑制剂抵消，见图3。 2.4 免疫荧光检测各组细胞的神经元早期标志物Ⅲβ-tubulin表达在诱导分化第12天，Ⅲβ-tubulin表达几乎贯穿整个神经元。与阴性对照组相比，miR-20 mimic组Ⅲβ-tubulin表达显著上调，但miR-20 inhibitor组和通路抑制剂组Ⅲβ-tubulin蛋白水平明显下调。联合组骨形态发生蛋白通路抑制剂能中和miR-20对Ⅲβ-tubulin生成的诱导，见图4。 2.5 各组高尔基染色结果为评定神经元发育及神经传递功能，通过高尔基染色观察各组人乳牙牙髓干细胞诱导的神经元树突棘密度。与阴性对照组相比，miR-20 mimic组单位面积树突棘数目显著增加，但miR-20 inhibitor组和通路抑制剂组树突棘稀疏。联合组骨形态发生蛋白通路抑制剂能中和miR-20对于神经元树突棘的诱导，见图5。"

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