Abstract
The aim of this study is to investigate the role of miR-9 and its mechanism on the osteoblast differentiation of mesenchymal stem cells. Real-time PCR and western blotting were used to study gene expression. Assay of Alkaline phosphatase activity and alizarin red staining were used to examine osteoblast differentiation. Transfection of miR-9 mimics or lent-shmiR-9 was used to modulate the level of miR-9 in C2C12. Overexpression of miR-9 in C2C12 cells stimulated alkaline phosphatase activity and osteoblast mineralization, as well as the expression of osteoblast marker genes Col I, Ocn and Bsp. Gene silencing of miR-9 in C2C12 resulted in the suppression of alkaline phosphatase activity and osteoblast mineralization, as well as the expression of Col I, Ocn and Bsp. DKK1 mRNA was not affected by miR-9 overexpression, however, DKK1 protein was significantly decreased. Moreover, DKK1 3′-UTR mediated transcriptional luciferase activity was also significantly suppressed by miR-9 overexpression. DKK1 mRNA was not affected by miR-9 gene silencing, however, DKK1 protein was significantly stimulated. Moreover, DKK1 3′-UTR mediated transcriptional luciferase activity was significantly stimulated by miR-9 gene silencing, and suppressed by miR-9 overexpression, however, DKK1 3′-UTR mutant mediated luciferase activity was unaffected. The siRNA derived gene silencing of DKK1 blocked the inhibiting effect of shmiR-9 on the expression of alkaline phosphatase; and blocked the inhibiting effect of shmiR-9 on the expression of ColI, Ocn and Bsp. MiR-9 promotes osteoblast differentiation of mesenchymal cell C2C12 by suppressing the gene expression of DKK1.
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Xiangyun Liu and Hao Xu contributed equally to this work.
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Liu, X., Xu, H., Kou, j. et al. MiR-9 promotes osteoblast differentiation of mesenchymal stem cells by inhibiting DKK1 gene expression. Mol Biol Rep 43, 939–946 (2016). https://doi.org/10.1007/s11033-016-4030-y
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DOI: https://doi.org/10.1007/s11033-016-4030-y