H19 Promotes Odontogenic Differentiation of Human Dental Pulp Cells via miR-103a-3p-Mediated PIK3R1/AKT and KLF4 Pathways

Background: The regeneration of functional dentin is a critical clinical goal for preserving tooth vitality after injury, with odontoblastic differentiation of human dental pulp stem cells (hDPSCs) being central to this reparative process. While the long noncoding RNA H19 is recognised as a key regulator of dentin repair, its downstream regulatory network is complex and incompletely mapped. Beyond the previously established H19/miR-140-5p/BMP2 axis, this study identifies a distinct and parallel pathway in which H19 promotes odontoblastic differentiation by downregulating miR-103a-3p, which in turn targets the PIK3R1/AKT and KLF4 signalling cascades. Our findings reveal that these two downstream networks operate independently, further elucidating the multifaceted role of H19 in dentin regeneration. Methodology: In this study, we used lentiviral vectors to stably overexpress H19 in hDPSCs. Bioinformatic analysis and dual-luciferase reporter assays were employed to validate the interactions between H19 and miR-103a-3p, as well as between miR-103a-3p and its target mRNAs, including phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and Kruppel-like factor 4 (KLF4). And qPCR and Western blot were used to investigate the expression pattern of H19 and the potential signalling axis of H19 and key odontogenic markers. Then, alkaline phosphatase and alizarin red S staining were used to evaluate odontogenic differentiation capacity. Finally, a heterotopic pulp regeneration model was established. And HE staining, Masson staining, immunofluorescence and immunohistochemistry were performed to verify the mechanism of H19 regulating odontogenic differentiation in vivo. Results: In vitro, H19 promoted odontogenic differentiation of hDPSCs, while miR-103a-3p inhibited them. Both PIK3R1 and KLF4 were identified as direct targets of miR-103a-3p. Ectopic expression of either PIK3R1 or KLF4 restored the odontogenic differentiation capacity of hDPSCs suppressed by miR-103a-3p. Mechanistically, PIK3R1 promoted odontogenesis by activating the PI3K/AKT signalling pathway, whereas KLF4 acted independently as a transcriptional regulator. In vivo, H19 overexpression drove odontoblastic differentiation of hDPSCs by inducing the expression of its downstream targets, PIK3R1 and KLF4. Conclusion: Our findings indicated that H19 promoted odontogenic differentiation of hDPSCs by modulating the miR-103a-3p-PIK3R1/AKT and miR-103a-3p-KLF4 axes, underscoring their therapeutic potential for pulp regeneration.