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

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...

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

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.