MicroRNA-224 suppresses osteoblast differentiation by inhibiting SMAD4

Osteoblast differentiation was found to be regulated by a variety of cell signaling and intracellular regulatory factors. In this study, we aimed at investigating the regulatory effect of microRNA-224 on osteoblast differentiation and its molecular mechanism. Expression of miR-224 in the osteoblasts, adipose-derived mesenchymal stem cells (MSC-A), bone marrow-derived mesenchymal stem cells (MSC-B) and mbilical cord-derived mesenchymal stem cells (MSC-U) were detected using RT-PCR. Expression of miR-224 was lower in osteoblast than in the three mesenchymal stem cells and it revealed a decreasing time-dependent trend from 0 day to 28 days during osteoblast differentiation. By using alkaline phosphatase (ALP) activity assay and alizarin red S (ARS) staining, we found that the mineralization nodules decreased in miR-224-mimics group and increased in miR-224-inhibitor group. The Western blot detection of osteoblast markers, such as osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), and runt related transcription factor 2 (RUNX2), also verified that overexpression of miR-224 inhibited osteoblast differentiation, while its inhibition promoted osteoblast differentiation. Luciferase reporter assay was performed in our study, which illustrated that miR-224 regulated SMAD4 directly by targeting SMAD4 3'UTR. Then after the inhibition of SMAD4, we found that lower expression of SMAD4 suppressed the osteoblast differentiation and the related signaling pathway using RT-PCR and Western blot. Our results revealed a new mechanism of osteoblast differentiation, and provided a new therapeutic agent to promote bone anabolism by targeting miR-224.