Abstract: BACKGROUND: Previous studies have confirmed that miR-27b at the lipid level can control multiple genes that have an important impact on dyslipidemia. It is speculated that miR-27b may function in osteoporosis by targeting peroxisome proliferators-activated receptors γ2 (PPARγ2).
OBJECTIVE: To investigate the effect of miR-27b/PPARγ2 axis in the dexamethasone-induced osteoporosis model in mouse embryonic osteogenic precursor cells MC3T3-E1 and related mechanisms of action.
METHODS: After MC3T3-E1 cells were dexamethasone-induced and cultured in vitro, miR-27b mimic, miR-27b inhibitor, NC-mimic, NC-inhibitor, siPPARγ2, and siNC 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 mRNA 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. Bioinformatics analysis predicted the downstream target genes of miR-27b followed by verification with dual luciferase gene reporter experiments.
RESULTS AND CONCLUSION: 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. 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. miR-27b overexpression attenuated the inhibitory effect of dexamethasone on the proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts. Dexamethasone significantly inhibited osteoblast differentiation compared with the dimethyl 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. Inhibition 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, osteoblast differentiation, alkaline phosphatase activity, and expression levels of bone morphogenetic protein 2, Runx2 and osteocalcin. PPARγ2 may be a direct target of miR-27b.

Key words: miR-27b, PPARγ2, MC3T3-E1, Runx2, osteogenic differentiation, proliferation, osteoporosis, dexamethasone