Department of Spinal Surgery, The Sixth Affiliated Hospital of Xinjiang Medical University, Urumqi City, Xinjiang, 830000, Uygur Autonomous Region, China.
Department of Endoscopy Center, The Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu Province, 215000, China.
In Vitro Cell Dev Biol Anim. 2021 Mar;57(3):350-358. doi: 10.1007/s11626-021-00553-x. Epub 2021 Mar 21.
This study aims to investigate the inhibitory effect of microRNA-337 (miR-337) on osteogenic differentiation in bone marrow mesenchymal stem cells and its action of mechanisms. Overexpression and knockdown of miR-337 were performed in bone marrow mesenchymal stem cells (BMSCs). Cell proliferation was assessed by using a cell counting kit-8 (CCK-8), mineralization assay was performed by alizarin red staining, and alkaline phosphatase activity was then measured. Luciferase reporter assay was applied to verify miR-337 binding to Ras-related protein 1A (Rap1A) mRNA. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) was applied to measure the expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), bone morphogenetic protein (BMP2), and miR-337. Then the protein level of Rap1A was determined by western blot analysis. High glucose inhibited osteogenic differentiation but increased the level of miR-337. Overexpression of miR-337 inhibited osteogenic differentiation in high glucose-treated BMSCs, while the knockdown of miR-337 reversed this process. Luciferase reporter assay confirmed that the presumed pairing binding site of miRNA-337 was in the 3'-UTR of the Rap1A WT. In addition, the knockdown of Rap1A distinctly repressed osteogenic differentiation, which blocked the effect of miR-337-knockdown on osteogenic differentiation in high glucose-treated BMSCs. MiR-337 could repress osteogenic differentiation in high glucose-treated BMSCs directly targeting Rap1A, thus provide a potential therapeutic strategy for patients with diabetic osteoporosis in clinic.
本研究旨在探讨 microRNA-337（miR-337）对骨髓间充质干细胞成骨分化的抑制作用及其作用机制。在骨髓间充质干细胞（BMSCs）中过表达和敲低 miR-337。使用细胞计数试剂盒-8（CCK-8）评估细胞增殖，通过茜素红染色进行矿化测定，然后测量碱性磷酸酶活性。应用荧光素酶报告实验验证 miR-337 与 Ras 相关蛋白 1A（Rap1A）mRNA 的结合。应用逆转录和定量聚合酶链反应（RT-qPCR）测量 Runt 相关转录因子 2（Runx2）、碱性磷酸酶（ALP）、骨钙素（OCN）、骨桥蛋白（OPN）、骨形态发生蛋白 2（BMP2）和 miR-337 的表达。然后通过 Western blot 分析测定 Rap1A 的蛋白水平。高葡萄糖抑制成骨分化，但增加 miR-337 的水平。在高葡萄糖处理的 BMSCs 中过表达 miR-337 抑制成骨分化，而敲低 miR-337 则逆转了这一过程。荧光素酶报告实验证实，miRNA-337 的假定配对结合位点位于 Rap1A WT 的 3'-UTR。此外，敲低 Rap1A 明显抑制成骨分化，阻断 miR-337 敲低对高葡萄糖处理的 BMSCs 中成骨分化的影响。MiR-337 可通过直接靶向 Rap1A 抑制高葡萄糖处理的 BMSCs 中的成骨分化，从而为临床糖尿病骨质疏松症患者提供一种潜在的治疗策略。
