Du Hurong, Zhang Wenlong, Liu Zeyuan, Liu Wei
Department of Orthopedics, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences，Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University , No. 99, Longcheng Street, Xiaodian District, Taiyuan, 030032, Shanxi, China.
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095, Jiefang Avenue, Wuhan, 430030, Hubei, China.
J Orthop Surg Res. 2025 Aug 7;20(1):741. doi: 10.1186/s13018-025-06159-3.
Fracture healing is a complex biological process involving precisely coordinated phases of inflammation, cartilage formation, vascularization, and bone remodeling. Growing evidence suggests that non-coding RNAs play crucial regulatory roles in bone metabolism.
This study systematically investigated the role and molecular mechanisms of lncRNA GABARAPL2 (lnc_GABARAPL2) in fracture healing through integrated bioinformatics analysis, clinical validation, and functional experiments, with the goal of identifying novel biomarkers and therapeutic targets for impaired fracture healing.
Lnc_GABARAPL2, miR-302a-3p, RUNX2 and OCN mRNA expression levels were quantified by RT-qPCR. Osteogenic differentiation was assessed through Western blot (Collagen I) and spectrophotometric ALP activity measurement and alizarin red S-based mineralization assay. In vitro, lnc_GABARAPL2 expression was modulated in hBMSCs via siRNA knockdown and overexpression vectors, with target interaction validated by dual-luciferase reporter assay. Cell proliferation (CCK-8) and apoptosis (flow cytometry) were assessed. Statistical methods included t-tests, ANOVA, and ROC curve analysis.
Lnc_GABARAPL2 expression was significantly elevated in non-union patients and demonstrated excellent predictive performance. Multivariate regression analysis confirmed lnc_GABARAPL2 as an independent predictor of non-union. Functional studies revealed that lnc_GABARAPL2 impaired osteogenic differentiation by suppressing ALP activity and reducing expression of key osteogenic markers RUNX2 and Collagen I, while modulating bone marrow mesenchymal stem cell proliferation and apoptosis through targeting miR-302a-3p.
These findings establish that lnc_GABARAPL2 may as a regulator of fracture healing and a promising predictive biomarker for non-union and targeting miR-302a-3p to modulate bone marrow mesenchymal stem cell functions, providing new insights for the development of RNA-based therapeutic strategies for impaired bone repair.
骨折愈合是一个复杂的生物学过程，涉及炎症、软骨形成、血管生成和骨重塑等精确协调的阶段。越来越多的证据表明，非编码RNA在骨代谢中发挥着关键的调节作用。
本研究通过综合生物信息学分析、临床验证和功能实验，系统地研究了lncRNA GABARAPL2（lnc_GABARAPL2）在骨折愈合中的作用和分子机制，旨在识别骨折愈合受损的新型生物标志物和治疗靶点。
通过RT-qPCR定量lnc_GABARAPL2、miR-302a-3p、RUNX2和OCN mRNA的表达水平。通过蛋白质印迹法（胶原蛋白I）、分光光度法测定碱性磷酸酶活性以及基于茜素红S的矿化试验评估成骨分化。在体外，通过siRNA敲低和过表达载体调节hBMSC中lnc_GABARAPL2的表达，并通过双荧光素酶报告基因试验验证靶标相互作用。评估细胞增殖（CCK-8）和细胞凋亡（流式细胞术）。统计方法包括t检验、方差分析和ROC曲线分析。
lnc_GABARAPL2在骨不连患者中的表达显著升高，并表现出优异的预测性能。多变量回归分析证实lnc_GABARAPL2是骨不连的独立预测因子。功能研究表明，lnc_GABARAPL2通过抑制碱性磷酸酶活性和降低关键成骨标志物RUNX2和胶原蛋白I的表达来损害成骨分化，同时通过靶向miR-302a-3p调节骨髓间充质干细胞的增殖和凋亡。
这些发现表明lnc_GABARAPL2可能作为骨折愈合的调节因子以及骨不连的有前景的预测生物标志物，并且靶向miR-302a-3p调节骨髓间充质干细胞功能，为开发基于RNA的骨修复受损治疗策略提供了新的见解。
