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 b...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.展开更多
基金National Natural Science Foundation of China(No.81860645)Hainan Medical University Introduced Talents Research Start-Up Funds(No.2015)。
文摘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.
