骨质疏松症患者骨髓干细胞来源的外泌体通过 microRNA-21/SMAD7 抑制成骨。
Bone marrow stem cells-derived exosomes extracted from osteoporosis patients inhibit osteogenesis via microRNA-21/SMAD7.
机构信息
Department of Orthopedics, Guangzhou University of Chinese Medicine, Guangzhou, China.
出版信息
Eur Rev Med Pharmacol Sci. 2018 Oct;22(19):6221-6229. doi: 10.26355/eurrev_201810_16028.
OBJECTIVE
To explore whether bone marrow stem cells (MSCs)-derived exosomes extracted from osteoporosis patients could inhibit osteogenesis via microRNA-21/SMAD7.
PATIENTS AND METHODS
MSCs from osteoporosis patients were isolated and cultured. MSCs morphology was observed, and the specific surface antigens were identified by flow cytometry. The osteogenic ability of MSCs was detected by alizarin red staining and oil red staining. Exosomes were collected from MSCs suspension by ultracentrifugation, and microRNA-21 expression in MSCs derived-exosomes was detected. Moreover, protein and mRNA levels of ALP, Bglap, and Runx2 in MSCs treated with different sources of MSCs-derived exosomes were detected by qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot, respectively. ALP activity in MSCs was accessed by a relative commercial kit. Furthermore, binding sites of microRNA-21 and SMAD7 were predicted by Targetscan, miRWalk, and miRDB, and were further verified by luciferase reporter gene assay. SMAD7 expression in MSCs derived-exosomes was also detected.
RESULTS
MSCs extracted from healthy adults, and osteoporosis patients were in adherent growth and exhibited elongated morphology, which could differentiate into osteoblasts and lipoblasts after different inductions. MicroRNA-21 expression in MSCs-derived exosomes extracted from osteoporosis patients was remarkably higher than those extracted from healthy adults. Decreased Runx2 expression and ALP activity were found after treatment of MSCs-derived exosomes extracted from osteoporosis patients. SMAD7 was confirmed to bind to microRNA-21 and was downregulated in osteoporosis patients in comparison with healthy adults. Overexpression of SMAD7 resulted in downregulated ALP, Bglap, and Runx2.
CONCLUSIONS
MicroRNA-21 inhibits osteogenesis through regulating MSCs-derived exosomes extracted from osteoporosis patients via targeting SMAD7.
目的
探讨骨质疏松症患者骨髓间充质干细胞（MSCs）来源的外泌体是否可以通过 microRNA-21/SMAD7 抑制成骨。
方法
分离培养骨质疏松症患者的 MSCs，观察 MSCs 形态，流式细胞术鉴定其表面特异性抗原。茜素红染色和油红染色检测 MSCs 的成骨能力。超速离心法从 MSCs 悬液中提取外泌体，检测 MSCs 来源外泌体中 microRNA-21 的表达。qRT-PCR（实时定量聚合酶链反应）和 Western blot 分别检测不同来源 MSCs 来源外泌体处理后的 MSCs 中碱性磷酸酶（ALP）、骨钙素（Bglap）和 runt 相关转录因子 2（Runx2）的蛋白和 mRNA 水平。用相对商业试剂盒检测 MSCs 中的 ALP 活性。进一步通过 Targetscan、miRWalk 和 miRDB 预测 microRNA-21 和 SMAD7 的结合位点，并通过荧光素酶报告基因检测进行验证。还检测了 MSCs 来源外泌体中的 SMAD7 表达。
结果
从健康成年人和骨质疏松症患者中提取的 MSCs 呈贴壁生长，呈长梭形，经不同诱导后可分化为成骨细胞和脂肪细胞。骨质疏松症患者 MSCs 来源外泌体中的 microRNA-21 表达明显高于健康成年人。骨质疏松症患者 MSCs 来源外泌体处理后 Runx2 表达和 ALP 活性降低。与健康成年人相比，骨质疏松症患者中 SMAD7 与 microRNA-21 结合并下调。过表达 SMAD7 导致 ALP、Bglap 和 Runx2 下调。
结论
microRNA-21 通过靶向 SMAD7 抑制骨质疏松症患者 MSCs 来源的外泌体，从而抑制成骨。
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