Abstract
OBJECTIVE: Fibronectin 1 (FN1) encodes fibronectin, a protein essential for cell adhesion, migration, extracellular matrix assembly, and regulation of cell differentiation and proliferation. While FN1 has been implicated in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), its role in neural differentiation remains unclear. This study aimed to investigate the effect of FN1 gene interference on neural differentiation of human BMSCs and explore the underlying molecular mechanisms. METHOD: Three small interfering RNAs (ssi-417, si-4467 and si-5468) targeting FN1 were designed and transfected into BMSCs undergoing neural differentiation. Morphological changes were observed, and FN1 expression was assessed at both mRNA and protein levels. Alkaline phosphatase (ALP) staining was performed, and the expression of neural differentiation-related markers (MAP2, Tuj1, NSE and DCX) was quantified. Transcriptome sequencing was used to identify differentially expressed genes (DEGs), alternative splicing (AS) events and key pathways. Protein-protein interaction (PPI) network analysis was conducted to identify hub genes. RESULT: Cells in the FN1 interference group retained a spindle-shaped mesenchymal morphology. FN1 expression at both mRNA and protein levels was significantly reduced in all three siRNA groups compared with the model group (P < 0.05). ALP staining showed a higher positive rate in the FN1 interference group. Expression of neural differentiation markers (MAP2, Tuj1, NSE and DCX) was significantly downregulated in the interference group compared with the model group (P < 0.05). Transcriptome analysis revealed 1047 upregulated and 1077 downregulated DEGs, enriched in pathways related to signal transduction, immune response, RNA processing, apoptosis and DNA repair. Additionally, 2246 alternative splicing events were identified, and PPI network analysis highlighted IL-6 as a core gene. CONCLUSION: FN1 gene interference inhibits neural differentiation of BMSCs and alters key signaling pathways and splicing patterns, suggesting that FN1 plays a critical role in regulating stem cell fate. These findings provide new insights into the molecular mechanisms underlying neural differentiation of BMSCs.