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

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

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。

PURPOSE: To study the role of microRNA(miR)-497-5p in the differentiation and mineralization of preosteoblasts 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, Smurf23’-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.