MicroRNA-431 effects on the differentiation and proliferation of human dental pulp stem cells

doi:10.3969/j.issn.2095-4344.0890

Abstract

Abstract:

BACKGROUND: Dental pulp stem cells have great clinical potential in dental repair and reconstruction through modern cellular and molecular biology and tissue engineering approaches.
OBJECTIVE: To explore the effect of microRNA-431 (miR-431) on the odontogenetic differentiation and in vitro proliferation of dental pulp stem cells.
METHODS: Dental pulp stem cells were isolated by enzyme digestion and induced to odontogenetic differentiation. Alkaline phosphatase activity was measured by Alkaline Phosphatase Assay Kit. Expression of odontogenetic differentiation markers, dentin sialophosphoprotein, osteocalcin and bone sialoprotein, was detected by western blot. Expression of miR-431 was detected by qRT-PCR. Cell proliferation and colony formation ability of dental pulp stem cells were detected by MTT and colony formation assay, respectively.
RESULTS AND CONCLUSION: Alkaline phosphatase activity and the expression of dentin sialophosphoprotein, osteocalcin and bone sialoprotein were increased but the expression of miR-431 reduced during the odontogenetic differentiation. Odontogenetic differentiation was inhibited by miR-431 overexpression, while miR-431-knockdown promoted the odontogenetic differentiation of dental pulp stem cells. These results indicate that miR-431 negatively regulates the odontogenetic differentiation of dental pulp stem cells, and it also inhibits the proliferation and colony formation ability of dental pulp stem cells.

Key words: dental pulp stem cells, odontogenetic differentiation, proliferation, clonal formation, stem cells

CLC Number:

Wu Wenhui, Guo Lei, Jiang Cuixia, Yang Yuling, Yuan Dongdong, Wang Nan. MicroRNA-431 effects on the differentiation and proliferation of human dental pulp stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3323-3328.

Figures/Tables 1

2.1 牙髓干细胞成牙分化过程中miR-431及成牙分化标志物的表达目前，临床上获取牙髓干细胞的方法是从自然替换的乳牙、智齿和畸牙等需要拔出的牙中收集牙髓，使用组织培养或酶消化等方法分离获得[22]。取健康前磨牙牙髓采用酶消化的方法成功分离培养出牙髓干细胞。为了检测牙髓干细胞成牙分化过程中miR-431及成牙分化标志物表达情况，提取出成牙分化前后的牙髓干细胞RNA，将提取的RNA反转录为cDNA，通过Real-time PCR检测miR-431表达。使用试剂盒和Western blot分别检测成牙分化前后碱性磷酸酶活性以及成牙分化标志蛋白牙本质涎磷蛋白(DSPP)、骨钙素(OCN)、骨唾液蛋白(BSP)的表达。结果显示，成牙分化诱导后碱性磷酸酶活性增加，成牙分化标志蛋白牙本质涎磷蛋白、骨钙素、骨唾液蛋白表达上升，miR-431表达量降低，提示miR-431负向调控成骨分化，见图1。 2.2 miR-431抑制牙髓干细胞的成牙分化为了检测miR-431对牙髓干细胞成牙分化的影响，实验将转染miR-431模拟剂或抑制剂的牙髓干细胞进行成牙分化诱导，培养12 d后检测miR-431表达、碱性磷酸酶活性以及成牙分化标志蛋白的表达。图2A的结果显示，转染miR-431模拟剂后细胞miR-431表达量明显升高，转染miR-431抑制剂后细胞miR-431表达显著降低。这些结果表明转染miR-431模拟剂和抑制剂可成功促进或者敲降牙髓干细胞miR-431的表达。图2B和图2C的结果显示，与正常分化的细胞相比，转染miR-431模拟剂后碱性磷酸酶活性降低，成牙分化标志蛋白表达降低，表明过表达miR-431成牙分化受到抑制。转染miR-431抑制剂后碱性磷酸酶活性增加，成牙分化标志蛋白表达上升，表明降低miR-431表达促进了成牙分化。 2.3 miR-431对牙髓干细胞增殖率的影响牙髓干细胞转染miR-431模拟剂和抑制剂，采用MTT法检测细胞增殖率，见图3。转染miR-431模拟剂后细胞增殖率下降，转染miR-431抑制剂后细胞增殖率上升，表明miR-431对牙髓干细胞的增殖有抑制作用。 2.4 miR-431对牙髓干细胞克隆形成能力的影响为了检测miR-431对牙髓干细胞克隆形成能力的影响，将牙髓干细胞培养12 d后显微镜下计数细胞克隆，计算克隆形成率，见图4。结果发现转染miR-431模拟剂后细胞集落形成数减少，转染miR-431抑制剂后集落形成增多，表明miR-431对牙髓干细胞集落形成有抑制作用。"

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