miR-483-5p regulates osteoclast generation by targeting Timp2.

Objective: To investigate whether miR-483-5p regulates osteoclast generation by targeting Timp2. miR-483-5p can promote osteoclast differentiation and bone destruction. Methods: Target genes of miR-483-5p were predicted by miRNAs target gene prediction software TargetScan8.0, and wild type and mutant 3' UTR plasmids were constructed. Dual luciferase reporter genes were used to verify whether target genes had a targeted regulatory relationship with miR-483-5p. Western blotting was used to detect the corresponding changes in the expression level of target protein after adjusting the level of miR-483-5p in cells. Cells were transfected or infected with target gene siRNA or target protein lentivirus, and TRAP staining and q-PCR assays were performed. In addition, for osteoclast induction experiment, RAW264.7 cells were co-transfected with ago-miR-483-5p and target protein-overexpressed lentiviruses q-PCR and TRAP staining were performed respectively. Results: Bioinformatics software was used to predict the target gene of miR-483-5p, and the Timp2 gene was found to regulate osteoclasts, and the dual luciferase reporter detection system found that miR-483- 5p could be associated with the 3-UTR of the predicted target gene Timp2 gene. There are complementary loci and targeted regulatory relationship between them. Subsequently, we upregulated miR-483-5p in RAW264.7 cells to reduce the expression of Timp2. Compared with the normal group, the number of osteoclasts and the expression of osteoclast-specific genes increased significantly after the induction of Timp2 in knockdown cells. After co-transfection of target gene and miR-483-5p into cells, the number of osteoclasts and the expression of specific genes decreased significantly compared with the normal group. Conclusion: Timp2 is a downstream target gene of miR-483-5p and is involved in and inhibits osteoclast generation. [ABSTRACT FROM AUTHOR]