The Effect of miR-129-5p on the Proliferation and Osteogenic Differentiation of Human Dental Pulp Stem Cells

Objective To investigate the role of microRNA-129-5p (miR-129-5p) in the proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Materials and Methods Differential expression analysis was performed using the GEO dataset GSE138180 to identify differentially expressed genes during the osteogenic differentiation of hDPSCs. Primary hDPSCs were isolated and cultured using the tissue block method combined with enzymatic digestion, and their stemness was characterized by alizarin red staining, oil red O staining, and flow cytometry. The expression dynamics of miR-129-5p at different stages of osteogenic induction were detected by quantitative real-time PCR (qRT-PCR). hDPSCs were transiently transfected with miR-129-5p mimic, negative control (mimic-NC), miR-129-5p inhibitor, or negative control (inhibitor-NC), and the transfection efficiency was verified by qRT-PCR. Cell proliferation was assessed by the CCK-8 assay. The expression of osteogenic-related genes (alkaline phosphatase [ALP], collagen type I [COL1], runt-related transcription factor 2 runt [RUNX2]) and proteins (ALP, osteopontin [OPN]) was measured using qRT-PCR, immunofluorescence, and Western blot, while mineral deposition was evaluated by alizarin red and alkaline phosphatase staining. Results Differential expression analysis revealed that miR-129-5p expression was significantly downregulated after 14 days of osteogenic induction compared with the control group. The primary cultured cells formed mineralized nodules and lipid droplets, were positive for CD90 and CD73, and were negative for CD45 and CD34. The expression of miR-129-5p continued to decrease after 7 and 14 days of osteogenic induction. Overexpression of miR-129-5p suppressed hDPSC proliferation, downregulated the expression of ALP, COL1, RUNX2, and OPN, and reduced mineral deposition. Conversely, inhibition of miR-129-5p promoted cell proliferation, enhanced the expression of osteogenic-related genes and proteins, and increased mineral deposition. Conclusion Inhibition of miR-129-5p promotes the proliferation and osteogenic differentiation of hDPSCs, suggesting that reduced miR-129-5p expression is associated with enhanced osteogenic potential and may have implications for pulp tissue engineering. Clinical Relevance This study investigated how miR-129-5p modulates the proliferation and osteogenic differentiation of hDPSCs. The findings elucidate its potential role in the regeneration of the pulp-dentin complex and indicate its therapeutic potential for dental tissue repair.