调控人骨髓间充质干细胞成骨分化的miR-106a及其靶基因骨形态发生受体蛋白2

摘要

背景：正常生理条件下，人骨髓间充质干细胞的骨生成和脂肪生成维持平衡，成骨分化是骨骼发展的重要过程，骨骼形成需要成骨细胞的分化和成熟。目的：探索miR-106及其靶基因骨形态发生受体蛋白2在成骨分化中的作用，为后续研究奠定基础。方法：使用成骨诱导培养基诱导骨髓间充质干细胞分化为成骨细胞，通过Western blot和碱性磷酸酶染色检测成骨分化标志物表达情况判断成骨分化进程。通过转染模拟剂过表达miR-106a，转染siRNA敲降骨形态发生受体蛋白2的表达，分别使用Real-time PCR和Western blot检测miR-106a和骨形态发生受体蛋白2表达情况。TargetScan软件预测和双荧光素酶报告实验验证miR-106a和骨形态发生受体蛋白2的相互作用。结果与结论：在成骨分化过程中miR-106a表达下调，骨形态发生受体蛋白2表达上调。过表达miR-106a，结果发现碱性磷酸酶活性下降，成骨分化标志蛋白Runx2和OCN表达量下降，同时骨形态发生受体蛋白2表达下调。使用TargetScan预测骨形态发生受体蛋白2可能是miR-106a的靶基因，双荧光素酶报告实验验证了预测结果。敲降骨形态发生受体蛋白2的表达，成骨分化受到抑制。结果表明miR-106a通过靶向骨形态发生受体蛋白2从而调控骨髓间充质干细胞向成骨细胞分化。%BACKGROUND:Under normal physiological conditions, there is a homeostasis between the osteogenic and adipogenic differentiation of human bone marrow mesenchymal stem cel s. Osteogenic differentiation is an important process in the formation of skeleton in which differentiated and mature osteoblasts are indispensable. OBJECTIVE:To explore the role of miR-106a and its target gene, bone morphogenetic protein receptor 2 (BMPR2) in the differentiation of human bone marrow mesenchymal stem cel s into osteoblasts. METHODS:Human bone marrow mesenchymal stem cel s were induced to differentiate into osteoblasts by osteoblast-specific induction medium, and this process was detected by western blot and alkaline phosphatase staining. Overexpression of miR-106a was elicited by transfecting miR-106a mimics and the BMPR2 knockdown achieved by RNA interference. The expression levels of miR-106a and BMPR2 were detected by real-time PCR and western blot, respectively. The interaction of miR-106a and BMPR2 was verified by TargetScan software and dual luciferase report experiment assay. RESULTS AND CONCLUSION:The expression of miR-106a was decreased whereas the expression of BMPR2 increased with the progress of osteogenesis differentiation. When miR-106a was overexpressed, alkaline phosphatase activity was declined and the expressions of runt-related transcription factor 2 and osteocalcin, markers of osteogenesis differentiation, both decreased. The expression of BMPR2 was decreased as wel . BMPR2 was predicted to be the target gene of miR-106a by TargetScan software and this prediction proved by dual luciferase report experiments assay. Additional y, osteogenesis differentiation was inhibited by knocking down the expression of BMPR2. These results indicate that miR-106a regulates the differentiation of bone marrow mesenchymal stem cel s into osteoblasts by targeting BMPR2.