Conclusions
Taken together, these data implied that miR-221 played an important part in osteoporosis through regulating RUNX2 expression and osteoblast differentiation.
Methods
Total RNA was extracted from fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 12) or osteoarthritis in the absence of osteoporosis (Control group, n = 12). Gene expression was quantified using TaqMan quantitative RT-PCR assays and protein production was determined by western blot analysis. The role of miR-221 in osteoblast differentiation was identified by gain or loss function experiment. MiRNA targets were identified using bioinformatics and luciferase reporter assay.
Results
MiR-221 was down-regulated in the osteoporotic samples compared with non-osteoporotic controls, and decreased in a C2C12 cell model of osteogenic differentiation. Overexpression of miR-221 resulted in a decrease in the osteogenic potential, as indicated by the reduced expression levels of key osteoblast markers, including osteocalcin (OC), alkaline phosphatase (ALP) and collagen, type I, α 1 (COL1A1), whereas inhibition of miR-221 promoted the activity of OC, ALP and COL1A1. Then bioinformatic analysis identified potential target sites of the miR-221 located in the 3' untranslated regions of RUNX2. Western blot analysis demonstrated that miR-221 inhibited RUNX2 gene expression. Furthermore, dual-luciferase reporter assays confirmed that RUNX2 was a direct target of miR-221. Rescue experiments showed that overexpression of RUNX2 significantly attenuated the effect of miR-221 on osteoblast markers providing strong evidence that miR-221 mediated the osteoblast differentiation by targeting RUNX2. Conclusions: Taken together, these data implied that miR-221 played an important part in osteoporosis through regulating RUNX2 expression and osteoblast differentiation.