Abstract
BACKGROUND: Fibronectin Leucine-Rich Transmembrane Protein 3 (FLRT3) plays a crucial role in craniofacial development and osteogenic differentiation, yet the regulatory mechanisms of microRNAs (miRNAs) on FLRT3 remain largely unexplored. This study identifies miR-144-3p as a miRNA that targets FLRT3 and investigates its role in osteogenic differentiation. These findings provide a foundation for the development of miRNA-based therapeutic strategies for craniofacial diseases and contribute to a deeper understanding of FLRT3's involvement in osteogenesis. METHODS: Mandibular bone marrow mesenchymal stem cells (MBMSCs) were isolated from SD rats using whole bone marrow and limiting dilution techniques. Flow cytometry and differentiation assays for osteogenesis and adipogenesis were conducted to characterize the cells. Bioinformatics analysis was employed to identify miRNAs that regulate FLRT3, with RT-qPCR used to select the most influential miRNA. A dual-luciferase reporter assay was performed to validate the interaction between the miRNAs and FLRT3. RT-qPCR measured miR-144-3p expression during osteogenic induction. Alkaline phosphatase and Alizarin Red S staining were used to assess the role of miR-144-3p in osteogenic differentiation. The impact of miR-144-3p on FLRT3, BMP2, and RUNX2 expression during osteogenesis was further evaluated by RT-qPCR. RESULTS: MBMSCs exhibited spindle-shaped morphology, high purity, and robust proliferative capacity, with flow cytometry confirming positive expression of CD29 and CD90. Bioinformatics analysis identified four miRNAs associated with FLRT3: miR-27a-3p, miR-27b-3p, miR-199a-3p, and miR-144-3p, with miR-144-3p showing the strongest correlation. Dual-luciferase assays confirmed that FLRT3 is a target gene of miR-144-3p. Functional assays revealed that the miR-144-3p mimic decreased FLRT3 expression, while the inhibitor increased it. During osteogenic differentiation, miR-144-3p mimic inhibited osteogenesis, while the inhibitor enhanced it, corresponding to alterations in the expression of FLRT3, BMP2, and RUNX2. CONCLUSION: miR-144-3p regulates the osteogenic differentiation of MBMSCs by targeting and modulating FLRT3, with FLRT3 being a direct target of miR-144-3p. During osteogenic differentiation, miR-144-3p inhibits osteogenesis by downregulating FLRT3, BMP2, and RUNX2 expression. This study not only elucidates the miRNA-mediated regulatory mechanism in osteogenesis but also offers a novel strategy for mandibular bone defect repair.