Abstract

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.