上海口腔医学

上海口腔医学 ›› 2023, Vol. 32 ›› Issue (1): 17-22.doi: 10.19439/j.sjos.2023.01.004

• 论著 • 上一篇    下一篇

miR-497-5p在前成骨细胞MC3T3-E1分化和矿化中的作用及机制探讨

胡熹1, 罗俊2   

  1. 1.南昌大学第二附属医院 口腔医学诊疗中心, 江西 南昌 330006;
    2.南昌大学附属口腔医院 正畸科, 江西省口腔生物医学重点实验室,江西 南昌 330006
  • 收稿日期:2021-11-30 修回日期:2022-01-18 出版日期:2023-02-25 发布日期:2023-06-12
  • 通讯作者: 罗俊,E-mail:174637426@qq.com
  • 作者简介:胡熹（1987-）,女,硕士研究生,主治医师,E-mail: 656605964@qq.com
  • 基金资助:
    江西省卫生健康委科技计划（SKJP20211554）; 江西省中医药管理局科技计划（2022A177）

Role and mechanism of miR-497-5p in the differentiation and mineralization of pre-osteoblast MC3T3-E1

HU Xi1, LUO Jun2   

  1. 1. Center of Stomatology, The Second Affiliated Hospital of Nanchang University. Nanchang 330006;
    2. Department of Orthodontics, Affiliated Stomatological Hospital of Nanchang University; Key Laboratory of Oral Biomedicine of Jiangxi Province. Nanchang 330006, Jiangxi Province, China
  • Received:2021-11-30 Revised:2022-01-18 Online:2023-02-25 Published:2023-06-12

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摘要： 目的: 研究微小RNA（miR）-497-5p在前成骨细胞MC3T3-E1分化和矿化中的作用,并探讨其相关机制。方法: 取第3代MC3T3-E1细胞,分别转染miR-497-5p过表达质粒miR-497-5p mimics、低表达质粒miR-497-5p inhibitor和阴性对照质粒miR-497-5p NC,设为miR-497-5p mimics组、miR-497-5p inhibitor组和miR-497-5p NC组。取不做处理的细胞作为空白组。成骨诱导后14 天,检测碱性磷酸酶（ALP）活性;免疫印迹法检测成骨分化相关蛋白骨钙素（OCN）、I型胶原（COL-I）蛋白表达量;茜素红染色法观察矿化情况;免疫印迹法检测Smad泛素化调节因子2（Smurf2）蛋白表达量;双荧光素酶实验验证miR-497-5p与Smurf2的靶向关系。采用SPSS 25.0软件包进行统计学分析。结果: 与空白组、miR-497-5p NC组相比,miR-497-5p mimics组ALP活性显著增强,OCN、COL-I蛋白表达量及矿化结节面积构成比显著升高,Smurf2蛋白表达量显著降低（P<0.05）;miR-497-5p inhibitor组ALP活性显著减弱,OCN、COL-I蛋白表达量及矿化结节面积构成比显著降低,Smurf2蛋白表达量显著升高（P<0.05）;与Smurf2 3’-UTR-WT+miR-497-5p NC组、Smurf2 3’-UTR-MT+miR-497-5p mimics组和Smurf2 3’-UTR-MT+miR-497-5p NC组相比,Smurf2 3’-UTR-WT+miR-497-5p mimics组双荧光素酶活性值显著降低（P<0.05）。结论: 过表达miR-497-5p对前成骨细胞MC3T3-E1的分化和矿化具有促进作用,其作用机制可能与负性靶向调控Smurf2蛋白表达有关。

关键词: 前成骨细胞, 微小RNA-497-5p, 分化, 矿化

Abstract: PURPOSE: To study the role of microRNA (miR)-497-5p in the differentiation and mineralization of pre-osteoblasts MC3T3-E1, and to explore the related mechanisms. METHODS: The third generation MC3T3-E1 cells were transfected into the miR-497-5p overexpression plasmid miR-497-5p mimics, the low expression plasmid miR-497-5p inhibitor, and the negative control plasmid miR-497-5p NC. They were set up as the miR-497-5p mimics group, miR-497-5p inhibitor group, and miR-497-5p NC group. The cells untreated was set up as the blank group. Fourteen days after osteogenic induction, alkaline phosphatase (ALP) activity was detected. The expression of osteocalcin (OCN) and type I collagen (COL-I) proteins related to osteogenic differentiation were detected by Western blotting. Mineralization was observed by alizarin red staining method. The expression of Smad ubiquitination regulatory factor 2 (Smurf2) protein was detected by Western blotting. The targeting relationship between miR-497-5p and Smurf2 was verified by dual luciferase experiment. Statistical analysis was performed by SPSS 25.0 software package. RESULTS: Compared with the blank group and miR-497-5p NC group, ALP activity of the miR-497-5p mimics group was enhanced, the expression of OCN, COL-I protein and the ratio of the area of mineralized nodules was increased, and the expression of Smurf2 protein was decreased(P<0.05). ALP activity of the miR-497-5p inhibitor group was weakened, the expression of OCN, COL-I protein and the ratio of the area of mineralized nodules was decreased, and the expression of Smurf2 protein was increased(P<0.05). Compared with Smurf2 3'-UTR-WT+miR-497-5p NC group, Smurf2 3'-UTR-MT+miR-497-5p mimics group, Smurf2 3'-UTR-MT+miR-497-5p NC group, the activity of dual luciferase in the WT+miR-497-5p mimics group was decreased (P<0.05). CONCLUSIONS: Overexpression of miR-497-5p can promote the differentiation and mineralization of pre-osteoblasts MC3T3-E1, and its mechanism may be related to the negatively targeted regulation of Smurf2 protein expression.

Key words: Preosteoblasts, MicroRNA-497-5p, Differentiation, Mineralization

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