MiR-296 Promotes Osteoblast Differentiation by Upregulating Cbfal.

We aim to identify the potential role of miR-296, which is a class of endogenous non-coding RNAs in regulating osteoblast differentiation. Human osteoblast cell line, hFOB 1.19 cells, were transfected with miR-296 overexpression or inhibition plasmid to upregulate or downregulate miR-296 expression, and then co-transfected with anti-Cbfal antibody to knockdown Cbfal. Alizarin red staining, alkaline phosphatase (ALP) activity, and enzyme-linked immunosorbent assay assays were performed to evaluate the extent of osteoblast differentiation. MTT assays were applied to measure cell proliferation. Wound healing and transwell assays were used to determine the ability of osteoblast migration and invasion. Flow cytometry assay was used to measure cell apoptosis and cell cycle change. The mRNA and protein expression of Cbfal, OSX, and Col-1 were confirmed by RT-qPCR and western blot respectively. We found that miR-296 overexpression contributed to a significant enhancement of matrix mineralization, ALP activity, and osteocalcin expression comparing with the negative control, whereas knockdown of miR-296 caused an opposite result. Meanwhile, the treatment of miR-296 promotes osteoblast migration, invasion, and proliferation, while it inhibits cell apoptosis. Compared with the negative control groups, the cells transfected with miR-296 also presented a much higher expression of Cbfal, OSX, and Col-1. Further experiments confirmed that the positive regulatory role of miR-296 in osteoblast differentiation is achieved by upregulating Cbfal expression. In conclusion, miR-296 promotes osteoblast differentiation by upregulating Cbfal expression in hFOB 1.19 cells, which may be used as a potential therapeutic target for the treatment of bone diseases in future.

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