Lu Xihong, Deng Min, He Honghui, Zeng Dehui, Zhang Wei
Department of Orthopedics, Affiliated Nanhua Hospital of University of South China, Hengyang Hunan,China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2013 Apr;38(4):341-6. doi: 10.3969/j.issn.1672-7347.2013.04.002.
To investigate whether miR-125b regulates the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) by modulating Smad4, a predicted target in silicon.
Smad4 3'-UTR-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-125b on luciferase activity. MSCs were isolated and cultured from human bone marrow, and then transfected with miR-125b mimics followed by induction of osteogenic differentiation. qRT-PCR and Western blot were used to detect the expressions of Smad4 mRNA and protein. MSCs were induced into the osteoblasts after transfecting with Smad4 siRNA, and the effect of Smad4 downregulation on osteogenic differentiation was observed by AKP activity and RUNX2 mRNA levels.
miR-216b bound Smad4 3'-UTR and inhibited the luciferase activity (P<0.05). Smad4 mRNA and protein expressions were significantly down-regulated in the MSCs induced into osteogenic differentiation when miR-125b was overexpressed. Downregulation of Smad4 suppressed the AKP activity and RUNX2 mRNA expression, indicating that Smad4 siRNA simulated at least in part the function of miR-125b as the regulator of MSCs osteogenic differentiation.
miR-125b can suppress MSCs osteogenic differentiation by directly targeting Smad4.
研究miR-125b是否通过调控Smad4（硅中的一个预测靶点）来调节骨髓间充质干细胞（MSCs）的成骨分化。
构建Smad4 3'-UTR荧光素酶载体，采用双荧光素酶报告基因检测法检测miR-125b对荧光素酶活性的影响。从人骨髓中分离培养MSCs，然后用miR-125b模拟物转染，随后诱导成骨分化。采用qRT-PCR和蛋白质印迹法检测Smad4 mRNA和蛋白的表达。用Smad4 siRNA转染MSCs后诱导其分化为成骨细胞，通过碱性磷酸酶（AKP）活性和RUNX2 mRNA水平观察Smad4下调对成骨分化的影响。
miR-216b与Smad4 3'-UTR结合并抑制荧光素酶活性（P<0.05）。过表达miR-125b时，诱导成骨分化的MSCs中Smad4 mRNA和蛋白表达显著下调。Smad4的下调抑制了AKP活性和RUNX2 mRNA表达，表明Smad4 siRNA至少部分模拟了miR-125b作为MSCs成骨分化调节因子的功能。
miR-125b可通过直接靶向Smad4抑制MSCs的成骨分化。
