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

doi:10.19439/j.sjos.2023.01.004

Abstract

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

CLC Number:

R681

HU Xi, LUO Jun. Role and mechanism of miR-497-5p in the differentiation and mineralization of pre-osteoblast MC3T3-E1[J]. Shanghai Journal of Stomatology, 2023, 32(1): 17-22.

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