Study Of MiR-27b Regulates Dexamethasone Induced MC3T3-E1 Cell Proliferation And Osteogenic Differentiation By Targeting PPARγ2

OBJECTIVE:To investigate the effects and related mechanisms of miR-27b and PPARγ2 on dexamethasone-induced mouse embryonic osteoblast precursor cells.METHODS:After MC3T3-E1 cells were dexamethasone-induced and cultured in vitro,miR-27b mimic,miR-27b inhibitor,NC-mimic,NC-inhibitor,si PPARγ2,and si NC were transfected with Lipofectamine^?2000,and dimethyl sulfoxide was used as a control.After the cells were induced and cultured,the cell viability and alkaline phosphatase activity were detected to determine the osteogenesis and differentiation levels of the cells.The m RNA and protein expression levels of osteogenic differentiation genes miR-27B,PPARγ2,Runt-related transcription factor 2（Runx2）,bone morphogenetic protein 2 and osteocalcin were detected by real-time fluorescence quantitative PCR and western blot methods.Predicted the downstream target genes of miR-27b followed by verification with dual luciferase gene reporter experiments.RESULTS:（1）Dexamethasone treatment significantly reduced cell viability and miR-27b expression levels in MC3T3-E1 pre-osteoblasts.Compared with dimethyl sulfoxide, dexamethasone significantly inhibited MC3T3-E1 cell viability and downregulated miR-27b expression levels at 24 and 48 hours.（2）miR-27b could directly regulate PPARγ2.Compared with the corresponding NC-mimic,the miR-27b mimic significantly upregulated the expression level of miR-27b,while the miR-27b inhibitor significantly downregulated the expression level of miR-27b.Furthermore,PPARγ2 miRNA and protein expression levels were inhibited by the miR-27b mimic and significantly enhanced by the miR-27b inhibitor.（3）miR-27b overexpression attenuated the inhibitory effect of dexamethasone on the proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts.Dexamethasone significantly inhibited osteogenic differentiation compared with the dimethy sulfoxide+NC-mimic group.In contrast,the dexamethasone-mediated inhibition was reversed by the miR-27b mimic.Cellular alkaline phosphatase activity and bone morphogenetic protein 2,Runx2 and osteocalcin protein expression levels were partially restored.（4）Knockout of miR-27b suppressed proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts through the upregulation of PPARγ2.Knockout of miR-27b significantly increased PPARγ2 expression and decreased cell viability,differentiation,alkaline phosphatase activity,and expression levels of bone morphogenetic protein 2,Runx2 and osteocalcin.PPARγ2 may be a direct target of miR-27b.CONCLUSION:miR-27b inhibits the PPARγ2 pathway and promotes dexamethasone-induced proliferation and osteogenic differentiation of MC3T3-E1 cells,thereby improving the development of osteoporosis and providing a new idea for the treatment of glucocorticoid osteoporosis.