Liu Hongzhi, Su Hao, Wang Xin, Hao Wei
Department of Orthopaedics and Traumatology, The Affiliated Yaitai Yuhuangding Hospital of Qingdao University Medical College, Yantai, Shandong, China.
J Cell Biochem. 2019 Feb;120(2):1350-1361. doi: 10.1002/jcb.27121. Epub 2018 Oct 18.
The purpose of this study was to investigate the underlying molecular mechanisms of fracture healing mediated by bone marrow mesenchymal stem cells. Differentially expressed microRNAs in acutely injured subjects and healthy volunteers were screened by microarray analysis. The dual luciferase reporter system was used to verify whether insulin-like growth factor 1 (IGF1) was the direct target gene regulated by miR-148a. The expression level of miR-148a and IGF1 after osteogenic differentiation was detected by quantitative real-time polymerase chain reaction. Western blot was used to determine the protein expression of bone markers, including IGF1, runt-related transcription factor 2 (Runx2), osteocalcin, and osteopontin in rat bone marrow-derived mesenchymal stem cells. Alkaline phosphatase and alizarin red staining was used to detect alkaline phosphatase activity and calcium deposition. An animal fracture model was used for in vivo experiments. MiR-148a was highly expressed in acutely injured subjects compared with healthy volunteers, and IGF1 was a target of miR-148a. Moreover, compared with the negative control group, IGF1 messenger RNA expression was significantly increased in the miR-148a antagomir group. During osteogenic differentiation, the expression of IGF1, Runx2, osteocalcin, and osteopontin was higher in the miR-148a antagomir group than other groups. In vivo experiments further confirmed that upregulation of IGF1 enhanced fracture healing efficiently by decreasing callus width and area and improving bone mineral density, maximum load, stiffness, and energy absorption. It was proved that IGF1 was the direct target gene of miR-148a, and the use of rat bone marrow-derived mesenchymal stem cells with low expression of miR-148a could improve fracture healing by upregulating IGF1.
本研究的目的是探讨骨髓间充质干细胞介导骨折愈合的潜在分子机制。通过微阵列分析筛选急性损伤受试者和健康志愿者中差异表达的微小RNA。使用双荧光素酶报告系统验证胰岛素样生长因子1（IGF1）是否为受miR-148a调控的直接靶基因。通过定量实时聚合酶链反应检测成骨分化后miR-148a和IGF1的表达水平。采用蛋白质印迹法测定大鼠骨髓来源间充质干细胞中包括IGF1、 runt相关转录因子2（Runx2）、骨钙素和骨桥蛋白在内的骨标志物的蛋白表达。使用碱性磷酸酶和茜素红染色检测碱性磷酸酶活性和钙沉积。采用动物骨折模型进行体内实验。与健康志愿者相比，miR-148a在急性损伤受试者中高表达，且IGF-1是miR-148a的靶标。此外，与阴性对照组相比，miR-148a拮抗剂组中IGF1信使核糖核酸表达显著增加。在成骨分化过程中，miR-148a拮抗剂组中IGF1、Runx2、骨钙素和骨桥蛋白的表达高于其他组。体内实验进一步证实上调IGF1可通过减小骨痂宽度和面积以及提高骨矿物质密度、最大负荷、刚度和能量吸收有效地促进骨折愈合。证明IGF1是miR-148a的直接靶基因，使用miR-148a低表达的大鼠骨髓来源间充质干细胞可通过上调IGF1来促进骨折愈合。
