Department of Orthopedics, The Second Hospital of Jilin University, Changchun, China.
The Fourth Medical Center of PLA General Hospital, Beijing, China.
Cell Biochem Funct. 2020 Dec;38(8):1152-1160. doi: 10.1002/cbf.3585. Epub 2020 Oct 12.
MiR-140-5p is high expressed in normal fracture healing, but its specific role and mechanism in tissue-to-bone healing are rarely reported. Therefore, this study investigated the effects of miR-140-5p on tissue-to-bone healing. Clone formation experiment, flow cytometry, Alizarin Red S Staining and Oil Red O Staining were performed to investigate the biological characteristics of mouse embryonic bone marrow mesenchymal stem cells C3H10T1/2. MiR-140-5p mimic was transfected into osteogenic medium (OS)-treated C3H10T1/2 cells to investigate the effects of miR-140-5p on osteogenic differentiation. MiR-140-5p transgenic mouse model and the transgenic fracture model were established, and the effects of miR-140-5p on osteogenic differentiation, bone mineral density (BMD) and bone mass of bone tissues were detected by haematoxylin and eosin staining and computed tomography scan. The expressions of osteocalcin, differentiation-related genes (Runx2, ALP, Spp1 and Bglap3) and miR-140-5p were determined by quantitative real-time polymerase chain reaction. C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells, and its phenotypes including CD44, CD90.1 and Sca-1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR-140-5p promoted the expressions of differentiation-related genes and calcium deposition of OS-treated C3H10T1/2 cells. MiR-140-5p increased the expression of osteocalcin, BMD and bone mass and promoted bone healing of miR-140-5p-transgenic mice with fracture. MiR-140-5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing in mice. SIGNIFICANCE OF THE STUDY: C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells and its phenotypes including CD44, CD90.1 and Sca-1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR-140-5p promoted the expressions of differentiation-related genes and calcium deposition of osteogenic medium-treated C3H10T1/2 cells. MiR-140-5p increased the expression of osteocalcin and bone mineral density and bone mass and promoted bone healing of miR-140-5p-transgenic mice with fracture. Our results showed that miR-140-5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing in mice, which may be a therapeutic target for treating fractures and promoting bone healing.
miR-140-5p 在正常骨折愈合中高表达，但它在组织向骨愈合中的具体作用和机制很少有报道。因此，本研究探讨了 miR-140-5p 对组织向骨愈合的影响。通过克隆形成实验、流式细胞术、茜素红 S 染色和油红 O 染色来研究小鼠胚胎骨髓间充质干细胞 C3H10T1/2 的生物学特性。将 miR-140-5p 模拟物转染到成骨培养基 (OS) 处理的 C3H10T1/2 细胞中，以研究 miR-140-5p 对成骨分化的影响。建立了 miR-140-5p 转基因小鼠模型和转基因骨折模型，通过苏木精和伊红染色和计算机断层扫描检测 miR-140-5p 对成骨分化、骨矿物质密度 (BMD) 和骨组织骨量的影响。通过实时定量聚合酶链反应测定成骨分化、分化相关基因 (Runx2、ALP、Spp1 和 Bglap3) 和 miR-140-5p 的表达。C3H10T1/2 细胞表现出形成成骨、脂肪细胞的克隆分化能力，以及其表型包括 CD44、CD90.1 和 Sca-1，但不包括造血干细胞标志物 CD45。miR-140-5p 的过表达促进了 OS 处理的 C3H10T1/2 细胞中分化相关基因的表达和钙沉积。miR-140-5p 增加了骨钙素的表达、BMD 和骨量，并促进了 miR-140-5p 转基因骨折小鼠的骨愈合。miR-140-5p 促进了小鼠胚胎骨髓间充质干细胞的成骨分化和骨折后的愈合。研究意义：C3H10T1/2 细胞表现出形成成骨、脂肪细胞的克隆分化能力，以及其表型包括 CD44、CD90.1 和 Sca-1，但不包括造血干细胞标志物 CD45。miR-140-5p 的过表达促进了 OS 处理的 C3H10T1/2 细胞中分化相关基因的表达和钙沉积。miR-140-5p 增加了骨钙素的表达、BMD 和骨量，并促进了 miR-140-5p 转基因骨折小鼠的骨愈合。我们的结果表明，miR-140-5p 促进了小鼠胚胎骨髓间充质干细胞的成骨分化和骨折后的愈合，这可能是治疗骨折和促进骨愈合的治疗靶点。
