MicroRNA-210 promotes the proliferation and odontogenic differentiation of rat dental pulp stem cells

doi:10.3969/j.issn.2095-4344.1787

Abstract

Abstract:

BACKGROUND: MicroRNAs profile predicts that microRNAs-210 may play a role in the differentiation of dental pulp stem cells into dentin and osteogenesis, but the specific role remains unclear.
OBJECTIVE: To investigate the role of microRNA-210 in the proliferation and odontogenic differentiation of rat dental pulp stem cells.
METHODS: The study was approved by the Ethics Committee of the Second Affiliated Hospital of Henan University of Chinese Medicine, China. The dental pulp tissues were collected from the mandibles of five Sprague-Dawley rats, and the pulp stem cells were isolated and identified. The dental pulp stem cells were divided into five groups. The normal control group was not treated. The cells in the miR-210 mimic negative control group were transfected with the miR-210 mimic negative control. The cells in the miR-210 mimic group were transfected with the miR-210 mimic. The cells in the miR-210 inhibitor negative control group were transfected with the miR-210 inhibitor negative control. The cells in the miR-210 inhibitor group were transfected with the miR-210 inhibitor negative control. Real-time quantitative PCR was used to detect the expression of microRNA-210, alkaline phosphatase, dentin sialophosphoprotein, osteopontin, osteocalcin, dentin matrix phosphoprotein-1 and GPCR-kinase interacting protein-2 in cells. Western blot was used to detect the expression of dentin sialophosphoprotein, osteopontin, osteocalcin and dentin matrix phosphoprotein-1 in cells. Cell counting kit-8 was used to detect cell proliferation. Cell alkaline phosphatase activity was detected by colorimetry. Alizarin red staining was used to detect the ability of mineral synthesis.
RESULTS AND CONCLUSION: (1) The proliferation activity of miR-210 mimic group was significantly higher than that of the other four groups (P < 0.01), while the proliferation activity of miR-210 inhibitor group was significantly lower than that of the other four groups (P < 0.01). (2) The activity of alkaline phosphatase in the miR-210 mimic group was significantly higher than that in the other four groups (P < 0.01), while the activity of alkaline phosphatase in the miR-210 inhibitor group was significantly lower than that in the other four groups (P < 0.01). (3) The results of alizarin red staining showed that the surfaces of cells in the miR-210 mimic group were covered with dark red nodules of different sizes owing to calcium salt deposition, and the number of dark red calcium nodules was higher than that in the other four groups. In the miR-210 inhibitor group, dark red calcium nodules were scattered on the cell surface, and the number of these nodules was lower than that in the other four groups. (4) The expressions of microRNA-210, alkaline phosphatase, dentin sialophosphoprotein, osteopontin, osteocalcin, dentin matrix phosphoprotein-1 and GPCR-kinase interacting protein-2 mRNAs and dentin sialophosphoprotein, osteopontin, osteocalcin and dentin matrix phosphoprotein-1 proteins were highest in the miR-210 mimic group and lowest in the miR-210 inhibitor group among the five groups, and there were significant differences among the five groups (P < 0.01). To conclude, microRNA-210 can promote the proliferation and odontogenic differentiation of rat dental pulp stem cells.

Key words: dental pulp stem cells, microRNA, microR-210, odontogenic differentiation, cell proliferation, alkaline phosphatase activity, mineral synthesis ability

CLC Number:

Wang Yanling, Zuo Chunran, Wang Jing, Yang Kun, Wang Shouru, Zhang Xiaoping. MicroRNA-210 promotes the proliferation and odontogenic differentiation of rat dental pulp stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4004-4010.

Figures/Tables 2

2.1 SD大鼠牙髓干细胞的鉴定结果流式细胞术检测结果显示，细胞表面标志物CD45、CD34的阳性表达率为1.1%，0.5%，CD29、CD44、CD73的阳性表达率为98.7%，98.5%，99.3%，见图1。结果提示从SD大鼠牙髓组织中提取的细胞具有牙髓干细胞的特征。 2.2 miR-210对SD大鼠牙髓干细胞增殖活力的影响 CCK-8检测结果显示，miR-210模拟物组细胞的增殖活力高于其他4组，差异有显著性意义(P < 0.01)；miR-210抑制物组细胞的增殖活力低于其他4组，差异有显著性意义 (P < 0.01)；正常对照组、miR-210模拟物阴性对照组和miR-210抑制物阴性对照组间细胞增殖活力差异无显著性意义(P > 0.05)，提示miR-210可提高SD大鼠牙髓干细胞的增殖活力，见图2。 2.3 miR-210对SD大鼠牙髓干细胞碱性磷酸酶活性的影响 miR-210模拟物组细胞的碱性磷酸酶活性高于其他4组，差异有显著性意义(P < 0.01)；miR-210抑制物组细胞的碱性磷酸酶活性低于其他4组，差异有显著性意义(P < 0.01)；正常对照组、miR-210模拟物阴性对照组和miR-210抑制物阴性对照组间细胞碱性磷酸酶活性差异无显著性意义(P > 0.05)，提示miR-210可提高SD大鼠牙髓干细胞的碱性磷酸酶活性，见图3。 2.4 miR-210对SD大鼠牙髓干细胞成骨分化能力的影响茜素红染色结果显示，miR-210模拟物组细胞表面布满钙盐沉积形成的大小不等的暗红色结节，数量多于其他4组，miR-210抑制物组细胞表面的暗红色钙结节散在分布，数量少于其他4组，提示miR-210可提高SD大鼠牙髓干细胞的成骨分化能力，见图4。 2.5 miR-210对SD大鼠牙髓干细胞牙源性分化相关基因和蛋白表达的影响 RT-qPCR结果显示，miR-210模拟物组细胞中miR-210、ALP、DSPP、DMP1、GIT2、OPN mRNA和DSPP、DMP1、OPN、OCN蛋白的表达高于其他4组，差异有显著性意义(P < 0.01)；miR-210抑制物组细胞中miR-210、ALP、DSPP、DMP1、GIT2、OPN mRNA和DSPP、DMP-1、OPN、OCN蛋白的表达低于其他4组，差异有显著性意义(P < 0.01)；正常对照组、miR-210模拟物阴性对照组和miR-210抑制物阴性对照组间各基因与蛋白的表达差异无显著性意义(P > 0.05)，提示miR-210可促进SD大鼠牙髓干细胞的牙源性分化，还可以正向调控GIT2的表达，见图5。"

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