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LncRNA GABARAPL2 promotes non‑union long bone fractures by suppressing bone mesenchymal stromal cell osteogenesis
Hurong Du1,2, Wenlong Zhang1,2, Zeyuan Liu1,2
1Department 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.
Background:
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.
Objective:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
Long non-coding RNA GABARAPL2 (lnc_GABARAPL2) is elevated in fracture non-union patients and impairs bone healing by inhibiting osteogenic differentiation. Targeting lnc_GABARAPL2 offers potential for new RNA-based therapies for bone repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Fracture healing is a complex biological process regulated by multiple factors.
- Non-coding RNAs are increasingly recognized for their critical roles in bone metabolism and healing.
- Impaired fracture healing, particularly non-union, presents a significant clinical challenge.
Purpose of the Study:
- To investigate the role and molecular mechanisms of long non-coding RNA GABARAPL2 (lnc_GABARAPL2) in fracture healing.
- To identify lnc_GABARAPL2 as a potential biomarker for non-union.
- To explore therapeutic strategies targeting lnc_GABARAPL2 and its downstream effectors for impaired bone repair.
Main Methods:
- Quantification of lnc_GABARAPL2, miR-302a-3p, RUNX2, and OCN mRNA expression using RT-qPCR.
- Assessment of osteogenic differentiation via Collagen I expression, ALP activity, and mineralization assays.
- In vitro manipulation of lnc_GABARAPL2 in human bone marrow mesenchymal stem cells (hBMSCs) and evaluation of cell proliferation and apoptosis.
Main Results:
- Lnc_GABARAPL2 expression was significantly higher in non-union patients and served as an independent predictor of non-union.
- Lnc_GABARAPL2 suppressed osteogenic differentiation by reducing RUNX2 and Collagen I expression and inhibiting ALP activity.
- Lnc_GABARAPL2 modulated hBMSC proliferation and apoptosis by targeting miR-302a-3p.
Conclusions:
- Lnc_GABARAPL2 acts as a regulator in fracture healing and is a promising predictive biomarker for non-union.
- Targeting lnc_GABARAPL2 and miR-302a-3p can modulate bone marrow mesenchymal stem cell functions.
- These findings provide novel insights for developing RNA-based therapeutic strategies for impaired bone repair.
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