MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix

•We investigated the impact of miR-214 on Osx expression.•Direct binding of miR-214 to the Osx 3′ UTR was demonstrated by luciferase reporter assay.•The Osx 3′ UTR contains two miR-214 binding sites.•MiR-214 suppresses the osteogenic differentiation of C2C12 myoblast cells.

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. Bioinformatic analyses predicted that miR-214 would be a potential regulator of Osx. The direct binding of miR-214 to the Osx 3′ untranslated region (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 U2OS 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 osteogenic differentiation of C2C12 cells was enhanced by the downregulation of miR-214 expression, as measured by increased alkaline phosphatase activity and matrix mineralization. Taken together, these results 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.