Department of Orthopedic, The First Affiliated Hospital of Soochow University, Souzhou, Jiangsu, China.
Department of Orthopedic, Taicang Affiliated Hospital of Soochow University, Taicang, Jiangsu, China.
J Cell Physiol. 2018 Oct;233(10):6929-6937. doi: 10.1002/jcp.26596. Epub 2018 Apr 25.
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.
成骨细胞分化受多种细胞信号和细胞内调节因子调控。本研究旨在探讨 microRNA-224 对成骨细胞分化的调节作用及其分子机制。采用 RT-PCR 检测成骨细胞、脂肪间充质干细胞（MSC-A）、骨髓间充质干细胞（MSC-B）和脐带间充质干细胞（MSC-U）中 miR-224 的表达。结果显示，成骨细胞中 miR-224 的表达低于三种间充质干细胞，且在成骨细胞分化过程中，从第 0 天到第 28 天呈时间依赖性逐渐降低的趋势。通过碱性磷酸酶（ALP）活性测定和茜素红 S（ARS）染色实验发现，miR-224 模拟物组矿化结节减少，miR-224 抑制剂组矿化结节增加。成骨细胞标志物骨钙素（OCN）、骨桥蛋白（OPN）、骨涎蛋白（BSP）和 runt 相关转录因子 2（RUNX2）的 Western blot 检测也证实，miR-224 的过表达抑制成骨细胞分化，而其抑制促进成骨细胞分化。本研究进行的荧光素酶报告基因检测表明，miR-224 通过靶向 SMAD4 3'UTR 直接调控 SMAD4。抑制 SMAD4 后，通过 RT-PCR 和 Western blot 发现 SMAD4 低表达抑制成骨细胞分化及其相关信号通路。本研究揭示了成骨细胞分化的新机制，为通过靶向 miR-224 促进骨合成提供了新的治疗靶点。
