Jiang Yikun, Zhang Jun, Li Zhengwei, Jia Guoliang
Department of Orthopedics, The Second Hospital of Jilin University, Changchun, China.
Front Med (Lausanne). 2020 Dec 7;7:577578. doi: 10.3389/fmed.2020.577578. eCollection 2020.
Recent evidence has demonstrated that mesenchymal stem cells (MSCs) can release a large number of functionally specific microRNA (miRNA) microvesicles that play a role in promoting osteogenic differentiation, but the specific mechanism is not yet clear. Under such context, this study aims to elucidate the mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) promoting fracture healing in mice. We isolated and identified the BMSC-Exo. Bioinformatics analysis predicted high expression of miRNA in exosomes and verified the transfer of miR-25 in exosomes by immunofluorescence. Targeting relationship between miR-25 and Smad ubiquitination regulatory factor-1 (SMURF1) was predicted and verified by dual-luciferase reporter gene assay. Immunoprecipitation and protein stability assays were used to detect Runt-related transcription factor 2 (Runx2) ubiquitination and the effect of SMURF1 on Runx2 ubiquitination, respectively. The effect of miR-25 in BMSC-Exo on fracture healing in mice was assessed using X-ray imaging. alkaline phosphatase, alizarin red staining, EdU, CCK-8, and Transwell were used to evaluate the effects of exosomes transferred miR-25 on osteogenic differentiation, proliferation, and migration of osteoblasts. Bioinformatics analysis predicted that miR-25 expression in exosomes increased significantly. Moreover, the targeted regulation of SMURF1 by miR-25 was verified. SMURF1 inhibited Runx2 protein expression by promoting ubiquitination degradation of Runx2. Notably, miR-25 secreted by BMSC-Exo can accelerate osteogenic differentiation, proliferation, and migration of osteoblasts through SMURF1/Runx2 axis. Our results demonstrate that miR-25 in BMSC-Exo regulates the ubiquitination degradation of Runx2 by SMURF1 to promote fracture healing in mice.
最近的证据表明，间充质干细胞（MSCs）可以释放大量具有功能特异性的微小RNA（miRNA）微囊泡，这些微囊泡在促进成骨分化中发挥作用，但其具体机制尚不清楚。在此背景下，本研究旨在阐明骨髓间充质干细胞来源的外泌体（BMSC-Exo）促进小鼠骨折愈合的机制。我们分离并鉴定了BMSC-Exo。生物信息学分析预测外泌体中miRNA高表达，并通过免疫荧光验证了外泌体中miR-25的转移。通过双荧光素酶报告基因检测预测并验证了miR-25与Smad泛素化调节因子-1（SMURF1）之间的靶向关系。分别采用免疫沉淀和蛋白质稳定性检测来检测Runx相关转录因子2（Runx2）的泛素化以及SMURF1对Runx2泛素化的影响。使用X射线成像评估BMSC-Exo中的miR-25对小鼠骨折愈合的影响。采用碱性磷酸酶、茜素红染色、EdU、CCK-8和Transwell评估外泌体转移的miR-25对成骨细胞成骨分化、增殖和迁移的影响。生物信息学分析预测外泌体中miR-25表达显著增加。此外，验证了miR-25对SMURF1的靶向调控。SMURF1通过促进Runx2的泛素化降解来抑制Runx2蛋白表达。值得注意的是，BMSC-Exo分泌的miR-25可通过SMURF1/Runx2轴加速成骨细胞的成骨分化、增殖和迁移。我们的结果表明，BMSC-Exo中的miR-25通过SMURF1调节Runx2的泛素化降解，从而促进小鼠骨折愈合。
