Department of Orthopedics, Beijing Friendship Hospital Capital Medical University, Beijing 100050, P.R. China.
Department of Orthopedics, The Third Clinical Medical College of Inner Mongolia Medical University, Baotou 014010, P.R. China.
Biochem Cell Biol. 2021 Jun;99(3):296-303. doi: 10.1139/bcb-2020-0163. Epub 2020 Oct 15.
This study examined the hypothesis that the microRNA miR-138-5p reduces the osteodifferentiation of human bone mesenchymal stem cells (hBMSCs) by downregulating the expression of forkhead box C1 (FOXC1). For this, hBMSCs were separated from bone marrow and osteogenic induction medium was added to stimulate osteogenic differentiation. Flow cytometric analysis was applied to evaluate the expression of cell-surface antigens associated with hBMSCs, including CD29, CD44, CD90, CD45, and CD34. qRT-PCR assays and Western blot assays were used to measure the mRNA and protein expression of miR-138-5p, osteocalcin, runt-related transcription factor 2, bone sialoprotein, alkaline phosphatase (ALP), and FOXC1. ALP staining assays and Alizarin Red staining (ARS) assays were used to confirm osteogenic differentiation. We used a luciferase assay to test the interaction between miR-138-5p and FOXC1. We demonstrated that miR-138-5p is downregulated in osteogenic differentiated hBMSCs. Further, overexpression of miR-138-5p reduced the expression of markers for osteodifferentiation, ALP activity, and ARS activity. Furthermore, we showed that is a downstream target gene of miR-138-5p, and that knockdown of miR-138-5p improves the osteogenesis differentiation of hBMSCs by upregulating . The results from this study indicate miR-138-5p as a new target for osteogenic differentiation of hBMSCs and the treatment of bone defects.
微小 RNA miR-138-5p 通过下调叉头框蛋白 C1（FOXC1）的表达，减少人骨髓间充质干细胞（hBMSCs）的成骨分化。为此，我们从骨髓中分离出 hBMSCs，并添加成骨诱导培养基以刺激成骨分化。采用流式细胞术分析评估与 hBMSCs 相关的细胞表面抗原的表达，包括 CD29、CD44、CD90、CD45 和 CD34。qRT-PCR 检测和 Western blot 检测用于测量 miR-138-5p、骨钙素、 runt 相关转录因子 2、骨唾液蛋白、碱性磷酸酶（ALP）和 FOXC1 的 mRNA 和蛋白表达。ALP 染色和茜素红染色（ARS）检测用于确认成骨分化。我们采用荧光素酶报告基因检测 miR-138-5p 与 FOXC1 之间的相互作用。结果表明，miR-138-5p 在成骨分化的 hBMSCs 中下调。此外，miR-138-5p 的过表达降低了成骨分化标志物、ALP 活性和 ARS 活性的表达。此外，我们还表明 是 miR-138-5p 的下游靶基因，miR-138-5p 的下调通过上调 来改善 hBMSCs 的成骨分化。本研究结果表明 miR-138-5p 可作为 hBMSCs 成骨分化和骨缺损治疗的新靶点。
