MicroRNA-214 Suppresses Osteogenic Differentiation Of C2C12 Myoblast Cells By Targeting Osterix

Osterix(Osx) is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Osx-null mice, which exhibit a complete absence of bone formation and arrested osteoblast differentiation, die immediately after birth. However, our understanding of the regulatory mechanism of Osx expression remains poor. MicroRNAs(miRNAs) are a class of small non-coding RNAs that play pivotal roles in diverse biological processes, including the development, differentiation, proliferation, survival, and oncogenesis of cells and organisms. In this study, we aimed to investigate the impact of miRNAs on Osx expression, thus to understand the molecular mechanisms of osteoblast differentiation.Bioinformatic analyses predicted that miR-214 would be a potential regulator of Osx, and there are two binding sites of miR-214 on Osx 3’ untranslated region(3’ UTR), indicating that mi R-214 may be a potential regulator of Osx. The direct binding of miR-214 to the Osx 3’UTR was demonstrated by a luciferase reporter assay using a construct containing the Osx 3’UTR. Deletion mutant construction revealed that the Osx 3’UTR contained two miR-214 binding sites. MiR-214 expression was inversely correlated with Osx expression in Saos-2 and U2 OS cells. The forced expression of miR-214 in Saos-2 cells led to a reduction in the level of Osx protein. Moreover, the role of miR-214 in the osteogenic differentiation of C2C12 cells was investigated. We found that the expression of miR-214 was decreased during the osteogenic differentiation of C2C12 cells. In addition, we found that the osteogenic differentiation of C2C12 cells was enhanced by the downregulation of miR-214 expression, as measured by detecting the expression of the osteoblast marker genes including ALP(alkaline phosphatase, ALP), Col1a1(collagen type I, Col1a1), and OC(osteocalcin, OC) using real-time PCR as well as detecting ALP activity and matrix mineralization by ALP staining and Alizarin Red staining. Taken together, these results firstly indicate that miR-214 is a novel regulator of Osx, and that it plays an important role in the osteogenic differentiation of C2C12 cells as a suppressor. Our findings have enriched the studies of regulatory mechanisms of Osx and are helpful to elucidate the processes of bone metabolism. Especially, it may be helpful in the treatment of osteoporosis and bone related diseases.