Gong Xiaokang, Chen Xi, Meng Zhulong, Huang Jiehe, Jia Shunjie, Wu Weiqian, Li Lihong, Zheng Xin
Department of Orthopedics, Municipal Hospital Affiliated to Taizhou University, Taizhou City, China.
Department of Pharmacology, School of Medicine, Taizhou University, Taizhou City, China.
Tissue Eng Part A. 2025 Aug;31(15-16):1066-1079. doi: 10.1089/ten.tea.2024.0273. Epub 2024 Dec 24.
Senescence and osteogenic differentiation potential loss limited bone nonunion treatment effects of bone marrow-derived mesenchymal stem cells (BMSCs). MiR-100-5p/Lysine(K)-specific demethylase 6B (KDM6B) can inhibit osteogenesis, but their effects on bone union remain unclear. This study aims to investigate the effects of miR-100-5p/KDM6B on osteogenic differentiation and bone defects. Wild-type or microRNA 100 (miR-100) knockdown mice underwent critical-size defect (CSD) cranial surgery and collagen I/poly-γ-glutamic acid scaffold treatment. The crania was observed using microcomputed tomography, hematoxylin and eosin staining, Masson staining, alkaline phosphatase (ALP) staining, immunohistochemistry, and immunofluorescence. Primary-cultured BMSCs transfected with miR-100-5p mimic/inhibitor and KDM6B cDNA were evaluated for osteogenic differentiation using Alizarin Red staining, ALP activity detection, and Western blot analysis. Genetic transcription levels were detected using quantitative reverse transcription polymerase chain reaction. This study found that miR-100 depletion promotes defect healing in mouse calvaria, increases the proportion of new bone and osteoblasts in calvaria, and activates the expression of KDM6B and osteocalcin (OCN) proteins, promoting the transcription of bone morphogenetic protein-2, Runt-related transcription factor 2 (Runx2), OCN, and KDM6B, while methylation of lysine 27 on histone H3 (H3K27me3) decreased. Furthermore, miR-100-5p mimics suppressed osteogenic differentiation by inhibiting KDM6B with increased H3K27me3, ALP, Runx2, OCN, and osteopontin protein expression, while miR-100-5p inhibitors have opposite effects. Moreover, KDM6B can reverse miR-100-5p mimic effects. Notably, scaffolds carrying miR-100-5p mimics/inhibitors transfected BMSCs were placed in CSD mice and found that miR-100-5p inhibitors have a better effect on CSD healing and increase new bone without inflammatory cell infiltration. This study proved that miR-100-5p depletion promotes bone union and osteogenic differentiation of BMSCs via KDM6B/H3K27me3.
衰老和成骨分化潜能丧失限制了骨髓间充质干细胞（BMSCs）对骨不连的治疗效果。微小RNA-100-5p（miR-100-5p）/赖氨酸（K）特异性去甲基化酶6B（KDM6B）可抑制成骨作用，但其对骨愈合的影响尚不清楚。本研究旨在探讨miR-100-5p/KDM6B对成骨分化和骨缺损的影响。对野生型或微小RNA 100（miR-100）敲低小鼠进行临界尺寸颅骨缺损（CSD）手术及I型胶原/聚γ-谷氨酸支架治疗。使用微型计算机断层扫描、苏木精-伊红染色、Masson染色、碱性磷酸酶（ALP）染色、免疫组织化学和免疫荧光观察颅骨情况。对用miR-100-5p模拟物/抑制剂和KDM6B cDNA转染的原代培养BMSCs，采用茜素红染色、ALP活性检测和蛋白质印迹分析评估其成骨分化情况。使用定量逆转录聚合酶链反应检测基因转录水平。本研究发现，miR-100缺失可促进小鼠颅骨缺损愈合，增加颅骨中新骨和成骨细胞比例，激活KDM6B和骨钙素（OCN）蛋白表达，促进骨形态发生蛋白-2、 runt相关转录因子2（Runx2）、OCN和KDM6B的转录，同时组蛋白H3赖氨酸27（H3K27me3）甲基化减少。此外，miR-100-5p模拟物通过抑制KDM6B抑制成骨分化，导致H3K27me3、ALP、Runx2、OCN和骨桥蛋白表达增加，而miR-100-5p抑制剂则有相反作用。而且，KDM6B可逆转miR-100-5p模拟物的作用。值得注意的是，将携带miR-100-5p模拟物/抑制剂转染BMSCs的支架植入CSD小鼠体内，发现miR-100-5p抑制剂对CSD愈合效果更好，可增加新骨形成且无炎性细胞浸润。本研究证明，miR-100-5p缺失通过KDM6B/H3K27me3促进BMSCs的骨愈合和成骨分化。
