H19 promotes odontogenic differentiation of human dental pulp cells via miR-103a-3p-mediated PIK3R1/AKT and KLF4 pathways

H19 promotes odontogenic differentiation of human dental pulp cells via miR-103a-3p-mediated PIK3R1/AKT and KLF4 pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article H19 promotes odontogenic differentiation of human dental pulp cells via miR-103a-3p-mediated PIK3R1/AKT and KLF4 pathways Jingkun Zhang, Li Lin, Huixian Dong, Bingtao Wang, Guangwei Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7499703/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Functional regeneration of the dentin–pulp complex is essential for restoring tooth integrity after injury. Odontoblastic differentiation of dental pulp stem cells (DPSCs) plays a central role in reparative dentinogenesis. Although lncRNA H19 is known to regulate biomineralization, its downstream network remains unclear. This study identified miR-103a-3p as a novel downstream effector of H19 and investigated its regulatory network in DPSCs odontoblastic differentiation. Methods Interactions between H19 and miR-103a-3p as well as miR-103a-3p and targets mRNAs (PIK3R1 and KLF4), were validated via bioinformatic and dual luciferase reporter assays. Quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of H19 and its potential signal axis and odontogenic markers. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to evaluated odontogenic ability. Finally, a heterotopic pulp regeneration model was established to reveal the regulating effects of H19. Results H19 acted as a sponge for miR-103a-3p, which otherwise inhibited the expression of odontogenic markers. PIK3R1 and KLF4 were identified as direct targets of miR-103a-3p. Overexpression of either PIK3R1 or KLF4 rescued the odontogenic differentiation capacity of DPSCs suppressed by miR-103a-3p. Furthermore, PIK3R1 promoted odontogenesis by activating the PI3K/AKT signaling pathway, while KLF4 functioned as an independent transcriptional regulator. Finally, in a heterotopic pulp regeneration model, H19 overexpression enhanced the expression of PIK3R1 and KLF4 and promoted odontoblastic differentiation of DPSCs in vivo. Conclusion Our results suggest that the H19-mediated miR-103a-3p/PIK3R1/AKT and miR-103a-3p/KLF4 axes promote the odontogenic differentiation of DPSCs and are expected to serve as therapeutic targets for pulp regeneration. H19 Odontogenic differentiation Dental pulp stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Dental caries and pulpitis are common oral diseases that frequently lead to hard tissue damage and pulp inflammation 1 , 2 . As neural crest derivatives, dental pulp stem cells demonstrate substantial self-renewal and multilineage differentiation capacities 3 . Under injury or inflammatory conditions, DPSCs can differentiate into odontoblast-like cells and contribute to reparative dentin formation, thereby facilitating tissue repair and pulp regeneration 4 , 5 . Owing to these properties, DPSCs are considered ideal seed cells for regenerative endodontics 6 . Therefore, identifying effective strategies to induce their odontogenic differentiation is critical for achieving functional dental tissue regeneration. Increasing evidence indicates that long noncoding RNAs (lncRNAs) play essential regulatory roles in stem cell fate determination and lineage commitment 7 , 8 . A primary mechanism of lncRNAs is acting as competing endogenous RNAs (ceRNAs) that sponge specific miRNAs, thereby relieving repression on their target mRNAs 9 . This ceRNA network plays a crucial role in fine-tuning gene expression during cell differentiation 10 . H19, a maternally expressed and developmentally regulated lncRNA, has been reported to promote odontogenic differentiation and tissue regeneration in DPSCs 11 , 12 . However, the underlying regulatory network of its pro-odontogenic function remains unclear. Based on preliminary bioinformatics analysis, we identified miR-103a-3p as a potential interacting miRNA of H19. miR-103a-3p is a conserved miRNA implicated in the regulation of cell proliferation, differentiation, and metabolism, and previous studies have shown that it inhibits the osteogenic differentiation of mesenchymal stem cells 13 . Nevertheless, whether H19 promotes odontogenesis through a miR-103a-3p–mediated regulatory network remains to be elucidated. In this study, we demonstrate that H19 promotes odontogenic differentiation of DPSCs by functioning as a ceRNA to sequester miR-103a-3p. Functional experiments revealed that miR-103a-3p suppresses odontogenic differentiation by targeting PIK3R1, a PI3K/AKT pathway activator, and KLF4, a transcription factor involved in odontoblast maturation. Together, our findings reveal a novel H19-mediated regulatory network involving the miR-103a-3p/PIK3R1/AKT and miR-103a-3p/KLF4 axes, which governs odontogenic differentiation in DPSCs and may offer a potential target for enhancing dental pulp regeneration. Methods Cell culture Human DPSCs were procured from Oricell (China). Experiments used hDPSCs at passages 3–6. For osteo/odontogenic induction, cells were seeded in 6-well plates at 1 × 10⁵ cells per well and induced with α-MEM supplemented with 10% FBS, 50 mg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, USA). HEK293T cells were procured from Procell (China). ALP assay and Alizarin Red S staining After 7 days of induction for ALP staining or 14 days for ARS staining, hDPSCs were fixed in 4% paraformaldehyde for 30 min at room temperature. For ALP detection, cells were treated with a BCIP/NBT alkaline phosphatase color development kit (Beyotime, China) for 10 min, while ARS staining was performed using 4.2% Alizarin Red S solution (Solarbio, China) for 20 min. Both staining procedures were carried out at room temperature in the dark. Images were captured with a stereomicroscope (DMi1; Leica). RNA preparation and qRT-PCR The reverse transcription and PCR amplification of non-coding RNAs were performed according to our standard protocols, as previously described 11 . The primer sequences provided in Supplementary Table S1 . Western blot analysis Western blot analysis Proteins were extracted on ice with RIPA lysis buffer (ST507; Beyotime, China) supplemented with 1% protease inhibitor. Lysates were quantified and 20 µg of protein per sample was separated via 10% SDS-PAGE, then transferred to 0.45 µm PVDF membranes (Millipore, USA). After transfer, membranes were blocked for 1 h at 25°C, then incubated overnight at 4°C with primary antibodies against GAPDH (1:1000; Abcam, UK), DSPP (1:1000; Abcam), DMP-1 (1:1000; Abcam, UK), KLF4 (1:1000; Proteintech, China), and PIK3R1 (1:1000; Cell Signaling Technology, USA). After washing three times with TBST for 5 min each, membranes were exposed to HRP-conjugated goat anti-rabbit (1:1000; Beyotime, China) or goat anti-mouse (1:5000; Proteintech, China) secondary antibodies for 1 h at room temperature. Protein signals were detected using an enhanced chemiluminescence kit (Epizyme, China), and band intensities were quantified with ImageJ software. Protein expression was normalized to GAPDH. Cell transfection and infection Small interfering RNA (siRNA) plasmids targeting KLF4 and PIK3R1, pcDNA3.1-based overexpression plasmids (KLF4, PIK3R1), miR-103a-3p mimics, miR-103a-3p inhibitor, and their negative controls (NC) were synthesized by GenePharma (China). hDPSCs at 70–80% confluence were transfected with these constructs using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s protocol. For H19 overexpression, lentiviral vectors LV-H19 and LV-NC (Obio Technology, China) were used to infect hDPSCs at 40% confluence. In both transfection and infection experiments, total RNA was extracted 48 h post-treatment, and qRT-PCR was performed to verify gene expression changes before subsequent osteo/odontogenic induction. Dual luciferase reporter assay The psiCHECK-2 luciferase reporter plasmid carrying the full-length H19 sequence, the 3′ untranslated regions (UTRs) of PIK3R1 or KLF4, and their respective mutant variants was obtained from Obio Technology and IGE Biotechnology (China). Twenty-four hours before transfection, HEK293T cells were plated into 24-well plates at a density of 5 × 10⁴ cells per well. Co-transfection was then carried out with the designated psiCHECK-2 constructs together with either miR-NC or miR-103a-3p mimics, using Lipofectamine 3000 (Invitrogen, USA) in accordance with the manufacturer’s protocol. After 48 h, Renilla and Firefly luciferase signals were quantified with Synergy™ 2 SL machine. All assays were conducted in triplicate. Animal studies The work has been reported in line with the ARRIVE guidelines 2.0. All animal procedures were approved by the Laboratory Animal Ethics Committee of Guangdong Ruiyi Testing Co., Ltd. and conducted in accordance with its guidelines. Experiments were performed under general anesthesia. Mice were anesthetized with Zoletil® 50 (Virbac; a combination of tiletamine hydrochloride and zolazepam hydrochloride) administered intraperitoneally at 50 mg/kg (combined dose). Depth of anesthesia was verified by loss of the pedal withdrawal reflex. A total of ten BALB/c mice (5 weeks old) were randomly allocated into the NC and H19 groups using a computer-generated random number sequence. All surgical procedures and outcome assessments were performed in a randomized order, and mice were housed under specific pathogen-free (SPF) conditions with cages rotated weekly to minimize potential confounding factors. Investigators performing the surgical procedures were aware of group allocation, whereas those responsible for histological evaluation and data analysis were blinded to the assignments to reduce assessment bias. No signs of pain or distress were observed during or after the procedures, and no unexpected adverse events occurred. All animals survived until the scheduled sacrifice and were included in subsequent analyses. Briefly, 1 × 10⁶ human dental pulp stem cells (hDPSCs) transduced with either H19 lentivirus or a negative control (NC) were mixed with Cellmatrix Type I (Cellmatrix, Osaka, Japan) and loaded into 3-mm-thick human dentin slices. The constructs were implanted subcutaneously into the dorsal region of the mice. Animals were provided ad libitum access to food and water and euthanized by cervical dislocation eight weeks after implantation. Following decalcification, tissue samples were sectioned at 5 µm, dehydrated through a graded ethanol series, and subjected to hematoxylin–eosin (HE) or Masson’s trichrome staining, or processed for immunofluorescence. Histological evaluation was performed under a light microscope. Statistical analysis Data analysis was carried out using GraphPad Prism 9.0 (GraphPad, La Jolla, USA). Comparisons between two groups were made with two-tailed Student’s t -tests, and differences among more than two groups were assessed by one-way analysis of variance (ANOVA). All values are presented as the mean ± standard deviation (SD) from three independent experiments. The P value less than 0.05 was considered statistically significant. Results 1.H19 Functions as a ceRNA to Promote Odontogenic Differentiation by Sponging miR-103a-3p lncRNAs can act as miRNA sponges. Our previous study showed that H19 promotes odontogenic differentiation of DPSCs 11 . To further investigate the underlying regulatory network, we screened for key miRNAs potentially targeted by H19 using databases including lncBase, RegRNA, ENCORI, and RNAInter. Three candidate miRNAs were identified: miR-423-5p, miR-339-5p, and miR-103a-3p (Fig. 1A). Among them, miR-103a-3p showed the most significant downregulation during odontogenic induction, and was thus selected for further study (Fig. 1B). Dual-luciferase reporter assays confirmed that H19 binds to miR-103a-3p (Fig. 1C-D). We constructed an H19 overexpression lentiviral vector to study its role in regulating miR-103a-3p, and overexpression efficiency was confirmed by qRT-PCR (Fig. 1E-F). In DPSCs, the miR-103a-3p expression were markedly decreased by LV- H19, suggesting that H19 negatively regulates miR-103a-3p (Fig. 1G). To investigate miR-103a-3p's role, DPSCs were transfected with its inhibitor (Fig. 1H). After 14 days of mineralization induction, qRT-PCR and Western blot showed increased expression of odontogenic markers (DSPP, DMP1, RUNX2, ALP) compared to controls, indicating that miR-103a-3p suppresses odontogenic differentiation (Fig. 1I–J). Rescue experiments showed that co-transfecting LV-H19 with miR-103a-3p mimics partially blocked H19's promoting effect on odontogenic differentiation (Fig. 1K). Western blot and ARS staining results were consistent (Fig. 1L-M). In summary, these data show that H19 promotes odontogenic differentiation of DPSCs, partly by downregulating miR-103a-3p. 2. miR-103a-3p Directly Targets PIK3R1 and KLF4 To explore the downstream targets of the H19/miR-103a-3p axis in regulating differentiation, we used miWalk, Tarbase, miRDB, and TargetScan to predict miR-103a-3p target genes, identifying 109 candidates (Fig. 2A). KEGG pathway analysis revealed significant enrichment of these genes in the 'Signaling pathways regulating pluripotency of stem cells' pathway, with PIK3R1 and KLF4 identified as key genes in this pathway, also linked to mineralization (Fig. 2B). Western blot results confirmed that the protein levels of PIK3R1 and KLF4 were significantly upregulated at days 7 and 14 of mineralization induction, suggesting their involvement in dentinogenic differentiation (Fig. 2C). Western blot analysis confirmed that the protein levels of PIK3R1 and KLF4 were significantly upregulated on days 7 and 14 of mineralization induction, suggesting their involvement in dentinogenic differentiation (Fig. 2C). Potential binding sites of miR-103a-3p on PIK3R1 and KLF4 were predicted (Fig. 2D), and dual-luciferase assays confirmed that miR-103a-3p directly targets PIK3R1 and KLF4 (Fig. 2E). Furthermore, qRT-PCR and Western blot results showed that PIK3R1 and KLF4 expression was reduced in the miR-103a-3p mimics group and increased in the miR-103a-3p inhibitor group, compared to the NC group (Fig. 2F-I). In conclusion, miR-103a-3p directly targets and suppresses PIK3R1 and KLF4 expression. 3. PIK3R1 reverses miR-103a-3p effects through PI3K/AKT pathway activation To further investigate the role of PIK3R1, three specific siRNAs (si-PIK3R1-1, si-PIK3R1-2, and si-PIK3R1-3) were synthesized and transfected into DPSCs (Fig. 3A). Among them, si-PIK3R1-3 exhibited the highest knockdown efficiency and was selected for subsequent experiments. After 14 days of mineralization induction, the expression of odontogenic genes was significantly downregulated in the PIK3R1 knockdown group compared to the control. Consistently, ALP and ARS staining revealed markedly reduced ALP activity and mineralized nodule formation after PIK3R1 knockdown (Fig. 3B–D). These results indicate that PIK3R1 downregulation suppresses DPSC odontogenic differentiation. Conversely, transfection with the PIK3R1 overexpression plasmid enhanced odontogenic gene expression and differentiation in DPSCs (Fig. 3E–G). To verify whether miR-103a-3p inhibits odontogenic differentiation via PIK3R1, we performed rescue experiments. Transfection of miR-103a-3p mimics significantly reduced odontogenic gene expression, while co-transfection with PIK3R1 overexpression plasmid reversed this inhibition (Fig. 3H-I). Similarly, miR-103a-3p mimics suppressed ALP activity and matrix mineralization, and these negative effects were reversed by PIK3R1 overexpression (Fig. 3J). These results suggest that PIK3R1 partially blocks the impact of miR-103a-3p on DPSCs. Since PIK3R1 is a key component of the PI3K/AKT signaling pathway, which plays a crucial role in the odontogenic differentiation of DPSCs 14–16 , we investigated whether PIK3R1 exerts its effects through this pathway. Overexpression of PIK3R1 in DPSCs significantly increased the p-AKT/AKT ratio, as shown by Western blot analysis (Fig. 3J). To verify PI3K/AKT pathway involvement, cells were then treated with the pathway inhibitor LY294002, which reduced p-AKT levels and partially reversed PIK3R1-induced odontogenic differentiation (Fig. 3K). These results suggest that PIK3R1 alleviates miR-103a-3p–induced suppression of odontogenic differentiation, at least in part, through activation of the PI3K/AKT signaling pathway. 4. KLF4 partially reverses miR-103a-3p – mediated suppression in DPSCs We also explored the role of KLF4 in odontogenic differentiation of DPSCs. Three KLF4-specific siRNAs (si-KLF4-1, si-KLF4-2, and si-KLF4-3) were synthesized and transfected into DPSCs, with si-KLF4-1, the most efficient knockdown, selected for further experiments (Fig. 4A). After 14 days of mineralization induction, odontogenic gene expression, ALP activity, and mineralized nodule formation were significantly reduced in the KLF4 knockdown group (Fig. 4B-D). Conversely, overexpression of KLF4 did the opposite, indicating its promotive role in odontogenic differentiation (Fig. 4E–G). To determine whether KLF4 could counteract the inhibitory effects of miR-103a-3p, we conducted rescue experiments. miR-103a-3p mimics markedly suppressed odontogenic differentiation, whereas co-transfection with a KLF4 overexpression plasmid largely reversed these inhibitory effects (Fig. 4H–J), suggesting that KLF4 partially blocks the negative impact of miR-103a-3p on DPSCs. 5. H19 promotes the odontogenic differentiation of DPSCs in vivo DPSCs with stable H19 overexpression (LV-H19) or control (LV-NC) cells were seeded into tooth root slices and implanted subcutaneously into BALB/c nude mice for 8 weeks (Fig. 5A-B). After implantation, pulp-like tissue formed within the root canal in the LV-H19 group (Fig. 5C). qRT-PCR analysis of regenerated tissues showed significantly higher H19 expression, elevated levels of odontogenic markers (ALP, RUNX2, DSPP, DMP1), and increased expression of target genes PIK3R1 and KLF4 compared to controls (Fig. 5D-G). H&E staining confirmed dentin-like tissue formation adjacent to the dentin matrix, while Masson’s trichrome staining revealed greater collagen deposition in the LV-H19 group (Fig. 5H-I). Immunofluorescence further demonstrated that PIK3R1 and KLF4 were predominantly localized within the newly formed dentin-like tissue (Fig. 5J). Collectively, these results indicate that H19 upregulates PIK3R1 and KLF4 expression and enhances the odontogenic differentiation of DPSCs in vivo. Discussion Dental pulp regeneration aims to reconstruct a functional pulp–dentin complex by restoring pulp vitality and promoting dentin repair, ultimately enabling the recovery of tooth function 17 . Dental pulp stem cells (DPSCs) play a central role in this process, given their capacity to differentiate into odontoblast-like cells, repair hard dental tissues, and support pulp regeneration 17 – 19 . Understanding the molecular mechanisms governing DPSCs odontogenic differentiation is essential for optimizing regenerative endodontic strategies. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that participate in diverse cellular processes, including growth, pluripotency, and differentiation 20 – 22 . Our previous lncRNA-array analysis identified H19 as an upregulated transcript during odontogenic differentiation of DPSCs, and subsequent in vitro studies confirmed its promotive role 11 . To investigate its in vivo function, DPSCs overexpressing H19 (LV-H19) were implanted into tooth root segments and transplanted subcutaneously into nude mice. This treatment markedly enhanced dentin-like tissue formation and increased expression of odontogenic markers DSPP and DMP1, demonstrating that H19 promotes reparative dentinogenesis in vivo. Although H19 has been implicated in the osteogenic and odontogenic differentiation of DPSCs, its precise molecular mechanism remains unclear. According to the competing endogenous RNA (ceRNA) hypothesis, lncRNAs can act as molecular sponges for microRNAs, thereby preventing them from suppressing their target mRNAs 23 , 24 . For example, H19 promotes MSC survival and angiogenic potential by sponging miR-199a-5p and upregulating its target gene VEGFA 25 . In this study, bioinformatics analysis predicted that miR-103a-3p could bind to lncRNA H19. While miR-103a-3p has been reported to inhibit osteogenic differentiation of ADSCs 26 , its role in DPSCs remains poorly understood. We therefore examined the interaction between miR-103a-3p and H19 and assessed its effect on odontogenic differentiation. Our results showed that H19 negatively regulates miR-103a-3p, and dual-luciferase assays confirmed their direct binding via a specific MRE, consistent with the ceRNA mechanism. Functionally, inhibition of miR-103a-3p enhanced odontogenic differentiation of DPSCs, as evidenced by increased expression of ALP, RUNX2, DSPP, and DMP1, together with greater matrix mineralization. In rescue experiments, miR-103a-3p mimics partially reversed the pro-odontogenic effects of H19, supporting the conclusion that H19 facilitates odontogenic differentiation by sponging miR-103a-3p through a ceRNA regulatory network. The PI3K/AKT signaling pathway plays a critical role in regulating cell proliferation, differentiation, and migration 27 – 29 . Its core components include phosphoinositide 3-kinase (PI3K), phosphatidylinositol-3,4,5-trisphosphate (PIP3), phosphoinositide-dependent kinase-1 (PDK1), and protein kinase B (AKT) 30 . Previous studies have shown that this pathway is involved in the osteo/odontogenic differentiation of tooth-derived mesenchymal stem cells (MSCs) 31 – 34 . Bioinformatics analysis predicted that the 3′ untranslated region (3′UTR) of PIK3R1 contains a sequence complementary to the seed region of miR-103a-3p, suggesting direct targeting. Interestingly, PIK3R1 encodes p85α, a regulatory subunit of the PI3K complex. This subunit activates the PI3K/AKT pathway by stabilizing and recruiting the catalytic subunit p110 to receptor tyrosine kinases, thereby promoting PIP3 production and subsequent AKT phosphorylation 35 , 36 . We hypothesized that lncRNA H19 promotes odontogenic differentiation of DPSCs by sponging miR-103a-3p, thereby upregulating PIK3R1 and activating PI3K/AKT signaling. To test this, a dual-luciferase reporter assay confirmed direct binding between miR-103a-3p and PIK3R1. Consistently, miR-103a-3p mimics reduced PIK3R1 expression, whereas inhibitors increased it, indicating post-transcriptional suppression. We next assessed PIK3R1 function and found that its overexpression promoted odontogenic differentiation of DPSCs. In rescue experiments, co-transfection of PIK3R1 with miR-103a-3p mimics restored odontogenic gene expression compared with miR-103a-3p mimics alone. We further examined the relationship between PIK3R1 and PI3K/AKT signaling. As expected, PIK3R1 overexpression increased the p-AKT/AKT ratio, while treatment with an AKT pathway inhibitor reversed this effect and reduced odontogenic gene expression. Collectively, these findings demonstrate that H19 facilitates PIK3R1 expression by sponging miR-103a-3p, thereby enhancing PI3K/AKT signaling and promoting odontogenic differentiation in DPSCs. KLF4 is a key transcription factor regulating stem cell pluripotency and differentiation 37 – 42 . Previous studies have shown that KLF4 binds to the promoters of mineralization-related genes, such as Dmp1 and Sp7, to regulate the odontogenic differentiation of DPSCs 43 , 44 . In KLF4 knockout mouse models, Chen et al. observed enlarged pulp chambers and defective dentin mineralization, underscoring its essential role in dentin formation 45 . In this study, KLF4 was predicted and confirmed to be a direct target of miR-103a-3p. KLF4 overexpression promoted odontogenic differentiation, consistent with its reported positive role in odontogenesis. Rescue experiments further showed that KLF4 co-expression partially reversed the inhibitory effects of miR-103a-3p, indicating that H19 enhances odontogenic differentiation by upregulating KLF4 through miR-103a-3p sponging. Conclusion In summary, this study highlights the odontogenic role of H19 in DPSCs, primarily through two key pathways: the lncRNA-H19/miR-103a-3p/PIK3R1/AKT axis and the lncRNA-H19/miR-103a-3p/KLF4 axis (Fig. 6). These findings provide new insights into the molecular mechanisms underlying dentin differentiation of DPSCs and may offer potential therapeutic targets for future regenerative strategies. Abbreviations hDPSCs: Human dental pulp stem cells; lncRNA: Long non-coding RNA; ceRNA: Competing endogenous RNA; qRT-PCR: Quantitative real-time polymerase chain reaction; DSPP: Dentin sialophosphoprotein; DMP-1: Dental matrix protein-1; ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; PIK3R1: Phosphoinositide-3-Kinase Regulatory Subunit 1; KLF4: Krüppel-like factor 4; PI3K: Phosphoinositide 3-kinase; AKT: AKT serine/threonine kinase; MSCs: Mesenchymal stem cells; miRNA: MicroRNA; 3′UTR: 3′ untranslated region; NC: Negative control Declarations Acknowledgements Not applicable. Authors’ contributions JKZ and LL contributed equally to this work and share first authorship. QZJ designed and conceived the study. JKZ critically performed the experiments. JKZ, LL, HXD, and GWC analyzed the data. JKZ, JLZ, CHW and BTW performed the statistical analysis. JKZ drafted the manuscript. JKZ, LL, QZJ, GWC and XSC critically revised the manuscript. QZJ provided funding. All authors read and approved the final manuscript. Funding This work was supported by the Science and Technology Program of Guangdong Provincial Science and Technology Department (no. 2016ZC0134) and Guangdong Basic and Applied Basic Research Foundation (no. 2024A1515012741). Data availability All data supporting this study are available in the article and its Supplementary Materials. The original sequencing data have been provided in the supplementary files. Ethics approval and consent to participate Human dental pulp stem cells (hDPSCs) were purchased from Oricell (Shanghai, China). The original source (Oricell) has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. All animal experiments were reviewed, approved, and supervised by the Experimental Animal Ethics Committee of Ruiye Model Animal (Guangzhou) Biotechnology Co. Ltd (Approval No. RYEth-20241026574; Project title: Mechanism of long non-coding RNA H19 regulating dentin-oriented differentiation of human dental pulp stem cells; Date of approval: October 26, 2024). Consent for publication The manuscript has been approved by all authors. As this study did not involve human participants or individual human data, informed consent was not required. Artificial intelligence The authors declare that they have not used AI‑generated work in this manuscript. Competing interests The authors declare that they have no competing interests. References Lin, L. et al. circ_0002456/FUS interaction inhibits NF-κB signaling to attenuate DNA damage and inflammatory responses in hDPSCs. Stem Cell Res. Ther. 16 , 276 (2025). Cheng, L. et al. Expert consensus on dental caries management. Int. J. Oral Sci. 14 , 17 (2022). Yamada, Y., Nakamura-Yamada, S., Kusano, K. & Baba, S. 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Supplementary Files Primersequences.pdf SupplementarymaterialWesternblot.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7499703","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":525270420,"identity":"6fbee63c-ab04-4bb0-bb47-12ad5e755898","order_by":0,"name":"Jingkun Zhang","email":"","orcid":"","institution":"Affiliated Stomatology Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingkun","middleName":"","lastName":"Zhang","suffix":""},{"id":525270423,"identity":"b4347454-7749-4560-9bc5-0a43ab77b19d","order_by":1,"name":"Li Lin","email":"","orcid":"","institution":"Affiliated Stomatology Hospital of 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1","display":"","copyAsset":false,"role":"figure","size":652104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH19 Functions as a ceRNA to Promote Odontogenic Differentiation by Sponging miR-103a-3p.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eDatabases consistently predicted three miRNAs interacting with lncRNA H19. \u003cstrong\u003eB \u003c/strong\u003eRelative candidate miRNA expression after odontoblast induction for 14 days compared with NC groups. \u003cstrong\u003eC\u003c/strong\u003e The binding site of miR-103a-3p with H19 were predicted. \u003cstrong\u003eD\u003c/strong\u003e The Dual luciferase assay. \u003cstrong\u003eE\u003c/strong\u003e Fluorescence was observed under an inverted fluorescence microscope after transduction for 48 h. \u003cstrong\u003eF\u003c/strong\u003eThe expression levels of H19 were determined by qRT-PCR. \u003cstrong\u003eG \u003c/strong\u003eThe expression levels of miR-103a-3p after transduction with H19 lentivirus. \u003cstrong\u003eH\u003c/strong\u003e qRT-PCR analysis was performed to measure the levels of miR-103a-3p. \u003cstrong\u003eI-J\u003c/strong\u003e Effects of miR-103a-3p knockdown on the expression of odontogenic differentiation-related genes in DPSCs. \u003cstrong\u003eK-L\u003c/strong\u003e Relative mRNA and protein expression of mineralization-related genes in cells cotransfected with LV-H19 and miR-103a-3p mimics after odontogenic differentiation for 14 days. M ARS staining results in cotransfection experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 (Full-length blots were presented in Supplementary Figure S1A-B)\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/4fd1ff55cdac764ea0c564be.png"},{"id":93065428,"identity":"4d247179-3fa5-41df-b0db-a66308889a71","added_by":"auto","created_at":"2025-10-08 16:43:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":503207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-103a-3p Directly Targets PIK3R1 and KLF4.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eIntersection of the predicted results in the data plot. \u003cstrong\u003eB \u003c/strong\u003eThe enriched KEGG functional pathways. \u003cstrong\u003eC\u003c/strong\u003eTarget genes (PIK3R1 and KLF4) protein expression after odontoblast induction for 7days and 14 days. \u003cstrong\u003eD\u003c/strong\u003e The binding site of target genes (PIK3R1 and KLF4) with miR-103a-3p were predicted. \u003cstrong\u003eE\u003c/strong\u003eDual luciferase assay. \u003cstrong\u003eF-G\u003c/strong\u003e Relative mRNA and protein expression of target genes (PIK3R1 and KLF4) in cells transfected with miR-103a-3p mimics after odontogenic differentiation for 14 days. \u003cstrong\u003eH-I \u003c/strong\u003eRelative mRNA and protein expression of target genes (PIK3R1 and KLF4) in cells transfected with miR-103a-3p inhibitor after odontogenic differentiation for 14 days. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 (Full-length blots were presented in Supplementary Figure S1C-E)\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/19e31a85bfbe4259213da7ab.png"},{"id":93065261,"identity":"e807ba0a-e6fb-466c-9fa7-841142967fbd","added_by":"auto","created_at":"2025-10-08 16:43:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1227729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePIK3R1 reverses miR-103a-3p effects through PI3K/AKT pathway activation.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eKnockdownefficiency of PIK3R1 in DPSCs. \u003cstrong\u003eB-C \u003c/strong\u003eEffects of PIK3R1 knockdown on the expression of odontogenic differentiation-related genes in DPSCs. \u003cstrong\u003eD\u003c/strong\u003eALP and ARS staining results. \u003cstrong\u003eE\u003c/strong\u003e Overexpressionefficiency of PIK3R1 in DPSCs. \u003cstrong\u003eF-G\u003c/strong\u003e Effects of PIK3R1 overexpression on the expression of odontogenic differentiation-related genes in DPSCs. \u003cstrong\u003eH-I\u003c/strong\u003e Relative mRNA and protein expression of mineralization-related genes in cells co-transfected with miR-103a-3p mimics and PIK3R1 overexpression plasmid after odontogenic differentiation for 14 days. \u003cstrong\u003eJ\u003c/strong\u003e ALP and ARS staining results in co-transfection experiments. \u003cstrong\u003eK\u003c/strong\u003e Protein expression of p-AKT, AKT, DSPP and DMP-1 in cells co-transfected with PIK3R1 overexpression plasmid and after treated with PI3K/AKT inhibitor LY294002. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Full-length blots were presented in Supplementary Figure S1F-I)\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/975048969edd2444af46990f.png"},{"id":93065523,"identity":"08f45650-8512-4d46-a8c2-df6cd064cd95","added_by":"auto","created_at":"2025-10-08 16:44:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1064547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLF4 partially reverses miR-103a-3p-mediated suppression in DPSCs.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eKnockdownefficiency of KLF4 in DPSCs. \u003cstrong\u003eB-C \u003c/strong\u003eEffects of KLF4knockdown on the expression of odontogenic differentiation-related genes in DPSCs. \u003cstrong\u003eD\u003c/strong\u003e ALP and ARS staining results. \u003cstrong\u003eE\u003c/strong\u003eOverexpression efficiency of KLF4 in DPSCs. \u003cstrong\u003eF-G\u003c/strong\u003e Effects of KLF4overexpression on the expression of odontogenic differentiation-related genes in DPSCs. \u003cstrong\u003eH-I\u003c/strong\u003e Relative mRNA and protein expression of mineralization-related genes in cells co-transfected with miR-103a-3p mimics and KLF4 overexpression plasmid after odontogenic differentiation for 14 days. \u003cstrong\u003eJ\u003c/strong\u003e ALP and ARS staining results in co-transfection experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 (Full-length blots were presented in Supplementary Figure S1J-L)\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/b05d851317e4f1f1f30bd439.png"},{"id":93065454,"identity":"c7a27b8c-3049-4ba4-a1a7-a06c04e86567","added_by":"auto","created_at":"2025-10-08 16:43:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2748182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH19 promotes the odontogenic differentiation of DPSCs in vivo. A \u003c/strong\u003eSchematic of subcutaneous transplantation in nude mice. \u003cstrong\u003eB \u003c/strong\u003eVivo experiments. \u003cstrong\u003eC\u003c/strong\u003e General view of the tooth root slices before transplantation and after 8 weeks of subcutaneous transplantation. \u003cstrong\u003eD-G\u003c/strong\u003e Dentin blocks seeded with H19- or NC-transduced DPSCs were implanted subcutaneously into nude mice for 8 weeks. mRNA levels of odontogenic genes (ALP, RUNX2, DSPP, DMP-1), H19 and target genes (PIK3R1 and KLF4) were analyzed by qRT-PCR. \u003cstrong\u003eH \u003c/strong\u003eRepresentative images of H\u0026amp;E staining of sections from tooth root slice. The new dentin-like structures are outlined by red dotted lines. Blue arrows: odontoblast-like cells; red arrows: blood vessels; nd: newly formed dentin; D: dentin. \u003cstrong\u003eI\u003c/strong\u003e Representative images of Masson’s trichrome staining of sections from tooth root slice.\u003cstrong\u003e J \u003c/strong\u003eImmunofluorescence staining for target genes (PIK3R1 and KLF4). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/9eb37d55e6c2218e25bae990.png"},{"id":93065344,"identity":"91b48c65-47b4-4549-822a-35852c4fd150","added_by":"auto","created_at":"2025-10-08 16:43:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":277414,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism diagram for H19-mediated miR-103a-3p/PIK3R1/AKT and miR-103a-3p/KLF4 axes.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/e8f6084fd903cf55ab46117a.png"},{"id":97670169,"identity":"49326bd4-a965-49f8-ac4b-545526e0f699","added_by":"auto","created_at":"2025-12-08 09:29:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7111170,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/9fe262e7-f588-4b49-9481-e45cf5752996.pdf"},{"id":93065457,"identity":"e233b848-4342-4ffe-a1b9-13002fbc054f","added_by":"auto","created_at":"2025-10-08 16:43:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":203895,"visible":true,"origin":"","legend":"","description":"","filename":"Primersequences.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/f44b0912ce4a88702ccda966.pdf"},{"id":93065341,"identity":"c386bf49-f921-4c28-b8fa-9591ae1648c3","added_by":"auto","created_at":"2025-10-08 16:43:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":38581482,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialWesternblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7499703/v1/0c903246b8aca19e4b87f6f2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"H19 promotes odontogenic differentiation of human dental pulp cells via miR-103a-3p-mediated PIK3R1/AKT and KLF4 pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDental caries and pulpitis are common oral diseases that frequently lead to hard tissue damage and pulp inflammation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As neural crest derivatives, dental pulp stem cells demonstrate substantial self-renewal and multilineage differentiation capacities\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Under injury or inflammatory conditions, DPSCs can differentiate into odontoblast-like cells and contribute to reparative dentin formation, thereby facilitating tissue repair and pulp regeneration\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Owing to these properties, DPSCs are considered ideal seed cells for regenerative endodontics\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, identifying effective strategies to induce their odontogenic differentiation is critical for achieving functional dental tissue regeneration.\u003c/p\u003e\u003cp\u003eIncreasing evidence indicates that long noncoding RNAs (lncRNAs) play essential regulatory roles in stem cell fate determination and lineage commitment\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A primary mechanism of lncRNAs is acting as competing endogenous RNAs (ceRNAs) that sponge specific miRNAs, thereby relieving repression on their target mRNAs\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This ceRNA network plays a crucial role in fine-tuning gene expression during cell differentiation\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. H19, a maternally expressed and developmentally regulated lncRNA, has been reported to promote odontogenic differentiation and tissue regeneration in DPSCs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the underlying regulatory network of its pro-odontogenic function remains unclear. Based on preliminary bioinformatics analysis, we identified miR-103a-3p as a potential interacting miRNA of H19. miR-103a-3p is a conserved miRNA implicated in the regulation of cell proliferation, differentiation, and metabolism, and previous studies have shown that it inhibits the osteogenic differentiation of mesenchymal stem cells\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Nevertheless, whether H19 promotes odontogenesis through a miR-103a-3p\u0026ndash;mediated regulatory network remains to be elucidated.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrate that H19 promotes odontogenic differentiation of DPSCs by functioning as a ceRNA to sequester miR-103a-3p. Functional experiments revealed that miR-103a-3p suppresses odontogenic differentiation by targeting PIK3R1, a PI3K/AKT pathway activator, and KLF4, a transcription factor involved in odontoblast maturation. Together, our findings reveal a novel H19-mediated regulatory network involving the miR-103a-3p/PIK3R1/AKT and miR-103a-3p/KLF4 axes, which governs odontogenic differentiation in DPSCs and may offer a potential target for enhancing dental pulp regeneration.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHuman DPSCs were procured from Oricell (China). Experiments used hDPSCs at passages 3\u0026ndash;6. For osteo/odontogenic induction, cells were seeded in 6-well plates at 1 \u0026times; 10⁵ cells per well and induced with α-MEM supplemented with 10% FBS, 50 mg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, USA). HEK293T cells were procured from Procell (China).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eALP assay and Alizarin Red S staining\u003c/h3\u003e\n\u003cp\u003eAfter 7 days of induction for ALP staining or 14 days for ARS staining, hDPSCs were fixed in 4% paraformaldehyde for 30 min at room temperature. For ALP detection, cells were treated with a BCIP/NBT alkaline phosphatase color development kit (Beyotime, China) for 10 min, while ARS staining was performed using 4.2% Alizarin Red S solution (Solarbio, China) for 20 min. Both staining procedures were carried out at room temperature in the dark. Images were captured with a stereomicroscope (DMi1; Leica).\u003c/p\u003e\n\u003ch3\u003eRNA preparation and qRT-PCR\u003c/h3\u003e\n\u003cp\u003eThe reverse transcription and PCR amplification of non-coding RNAs were performed according to our standard protocols, as previously described\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The primer sequences provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis\u003c/div\u003e\u003cp\u003eProteins were extracted on ice with RIPA lysis buffer (ST507; Beyotime, China) supplemented with 1% protease inhibitor. Lysates were quantified and 20 \u0026micro;g of protein per sample was separated via 10% SDS-PAGE, then transferred to 0.45 \u0026micro;m PVDF membranes (Millipore, USA). After transfer, membranes were blocked for 1 h at 25\u0026deg;C, then incubated overnight at 4\u0026deg;C with primary antibodies against GAPDH (1:1000; Abcam, UK), DSPP (1:1000; Abcam), DMP-1 (1:1000; Abcam, UK), KLF4 (1:1000; Proteintech, China), and PIK3R1 (1:1000; Cell Signaling Technology, USA). After washing three times with TBST for 5 min each, membranes were exposed to HRP-conjugated goat anti-rabbit (1:1000; Beyotime, China) or goat anti-mouse (1:5000; Proteintech, China) secondary antibodies for 1 h at room temperature. Protein signals were detected using an enhanced chemiluminescence kit (Epizyme, China), and band intensities were quantified with ImageJ software. Protein expression was normalized to GAPDH.\u003c/p\u003e\n\u003ch3\u003eCell transfection and infection\u003c/h3\u003e\n\u003cp\u003eSmall interfering RNA (siRNA) plasmids targeting KLF4 and PIK3R1, pcDNA3.1-based overexpression plasmids (KLF4, PIK3R1), miR-103a-3p mimics, miR-103a-3p inhibitor, and their negative controls (NC) were synthesized by GenePharma (China). hDPSCs at 70\u0026ndash;80% confluence were transfected with these constructs using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer\u0026rsquo;s protocol. For H19 overexpression, lentiviral vectors LV-H19 and LV-NC (Obio Technology, China) were used to infect hDPSCs at 40% confluence. In both transfection and infection experiments, total RNA was extracted 48 h post-treatment, and qRT-PCR was performed to verify gene expression changes before subsequent osteo/odontogenic induction.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDual luciferase reporter assay\u003c/h2\u003e\u003cp\u003eThe psiCHECK-2 luciferase reporter plasmid carrying the full-length H19 sequence, the 3\u0026prime; untranslated regions (UTRs) of PIK3R1 or KLF4, and their respective mutant variants was obtained from Obio Technology and IGE Biotechnology (China). Twenty-four hours before transfection, HEK293T cells were plated into 24-well plates at a density of 5 \u0026times; 10⁴ cells per well. Co-transfection was then carried out with the designated psiCHECK-2 constructs together with either miR-NC or miR-103a-3p mimics, using Lipofectamine 3000 (Invitrogen, USA) in accordance with the manufacturer\u0026rsquo;s protocol. After 48 h, Renilla and Firefly luciferase signals were quantified with Synergy\u0026trade; 2 SL machine. All assays were conducted in triplicate.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal studies\u003c/h3\u003e\n\u003cp\u003eThe work has been reported in line with the ARRIVE guidelines 2.0. All animal procedures were approved by the Laboratory Animal Ethics Committee of Guangdong Ruiyi Testing Co., Ltd. and conducted in accordance with its guidelines. Experiments were performed under general anesthesia. Mice were anesthetized with Zoletil\u0026reg; 50 (Virbac; a combination of tiletamine hydrochloride and zolazepam hydrochloride) administered intraperitoneally at 50 mg/kg (combined dose). Depth of anesthesia was verified by loss of the pedal withdrawal reflex. A total of ten BALB/c mice (5 weeks old) were randomly allocated into the NC and H19 groups using a computer-generated random number sequence. All surgical procedures and outcome assessments were performed in a randomized order, and mice were housed under specific pathogen-free (SPF) conditions with cages rotated weekly to minimize potential confounding factors. Investigators performing the surgical procedures were aware of group allocation, whereas those responsible for histological evaluation and data analysis were blinded to the assignments to reduce assessment bias. No signs of pain or distress were observed during or after the procedures, and no unexpected adverse events occurred. All animals survived until the scheduled sacrifice and were included in subsequent analyses.\u003c/p\u003e\u003cp\u003eBriefly, 1 \u0026times; 10⁶ human dental pulp stem cells (hDPSCs) transduced with either H19 lentivirus or a negative control (NC) were mixed with Cellmatrix Type I (Cellmatrix, Osaka, Japan) and loaded into 3-mm-thick human dentin slices. The constructs were implanted subcutaneously into the dorsal region of the mice. Animals were provided ad libitum access to food and water and euthanized by cervical dislocation eight weeks after implantation. Following decalcification, tissue samples were sectioned at 5 \u0026micro;m, dehydrated through a graded ethanol series, and subjected to hematoxylin\u0026ndash;eosin (HE) or Masson\u0026rsquo;s trichrome staining, or processed for immunofluorescence. Histological evaluation was performed under a light microscope.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData analysis was carried out using GraphPad Prism 9.0 (GraphPad, La Jolla, USA). Comparisons between two groups were made with two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests, and differences among more than two groups were assessed by one-way analysis of variance (ANOVA). All values are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent experiments. The \u003cem\u003eP\u003c/em\u003e value less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.H19 Functions as a ceRNA to Promote Odontogenic Differentiation by Sponging miR-103a-3p\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003elncRNAs can act as miRNA sponges. Our previous study showed that H19 promotes odontogenic differentiation of DPSCs\u003csup\u003e11\u003c/sup\u003e. To further investigate the underlying regulatory network, we screened for key miRNAs potentially targeted by H19 using databases including lncBase, RegRNA, ENCORI, and RNAInter. Three candidate miRNAs were identified: miR-423-5p, miR-339-5p, and miR-103a-3p (Fig. 1A). Among them, miR-103a-3p showed the most significant downregulation during odontogenic induction, and was thus selected for further study (Fig. 1B). Dual-luciferase reporter assays confirmed that H19 binds to miR-103a-3p (Fig. 1C-D). We constructed an H19 overexpression lentiviral vector to study its role in regulating miR-103a-3p, and overexpression efficiency was confirmed by qRT-PCR (Fig. 1E-F). In DPSCs, the miR-103a-3p expression were markedly decreased by LV- H19, suggesting that H19 negatively regulates miR-103a-3p (Fig. 1G). To investigate miR-103a-3p\u0026apos;s role, DPSCs were transfected with its inhibitor (Fig. 1H). After 14 days of mineralization induction, qRT-PCR and Western blot showed increased expression of odontogenic markers (DSPP, DMP1, RUNX2, ALP) compared to controls, indicating that miR-103a-3p suppresses odontogenic differentiation (Fig. 1I\u0026ndash;J). Rescue experiments showed that co-transfecting LV-H19 with miR-103a-3p mimics partially blocked H19\u0026apos;s promoting effect on odontogenic differentiation (Fig. 1K). Western blot and ARS staining results were consistent (Fig. 1L-M). In summary, these data show that H19 promotes odontogenic differentiation of DPSCs, partly by downregulating miR-103a-3p.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. miR-103a-3p Directly Targets PIK3R1 and KLF4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the downstream targets of the H19/miR-103a-3p axis in regulating differentiation, we used miWalk, Tarbase, miRDB, and TargetScan to predict miR-103a-3p target genes, identifying 109 candidates (Fig. 2A). KEGG pathway analysis revealed significant enrichment of these genes in the \u0026apos;Signaling pathways regulating pluripotency of stem cells\u0026apos; pathway, with PIK3R1 and KLF4 identified as key genes in this pathway, also linked to mineralization (Fig. 2B). Western blot results confirmed that the protein levels of PIK3R1 and KLF4 were significantly upregulated at days 7 and 14 of mineralization induction, suggesting their involvement in dentinogenic differentiation (Fig. 2C). Western blot analysis confirmed that the protein levels of PIK3R1 and KLF4 were significantly upregulated on days 7 and 14 of mineralization induction, suggesting their involvement in dentinogenic differentiation (Fig. 2C). Potential binding sites of miR-103a-3p on PIK3R1 and KLF4 were predicted (Fig. 2D), and dual-luciferase assays confirmed that miR-103a-3p directly targets PIK3R1 and KLF4 (Fig. 2E). Furthermore, qRT-PCR and Western blot results showed that PIK3R1 and KLF4 expression was reduced in the miR-103a-3p mimics group and increased in the miR-103a-3p inhibitor group, compared to the NC group (Fig. 2F-I). In conclusion, miR-103a-3p directly targets and suppresses PIK3R1 and KLF4 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. PIK3R1 reverses miR-103a-3p effects through PI3K/AKT pathway activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the role of PIK3R1, three specific siRNAs (si-PIK3R1-1, si-PIK3R1-2, and si-PIK3R1-3) were synthesized and transfected into DPSCs (Fig. 3A). Among them, si-PIK3R1-3 exhibited the highest knockdown efficiency and was selected for subsequent experiments. After 14 days of mineralization induction, the expression of odontogenic genes was significantly downregulated in the PIK3R1 knockdown group compared to the control. Consistently, ALP and ARS staining revealed markedly reduced ALP activity and mineralized nodule formation after PIK3R1 knockdown (Fig. 3B\u0026ndash;D). These results indicate that PIK3R1 downregulation suppresses DPSC odontogenic differentiation. Conversely, transfection with the PIK3R1 overexpression plasmid enhanced odontogenic gene expression and differentiation in DPSCs (Fig. 3E\u0026ndash;G). To verify whether miR-103a-3p inhibits odontogenic differentiation via PIK3R1, we performed rescue experiments. Transfection of miR-103a-3p mimics significantly reduced odontogenic gene expression, while co-transfection with PIK3R1 overexpression plasmid reversed this inhibition\u0026nbsp;(Fig. 3H-I). Similarly, miR-103a-3p mimics suppressed ALP activity and matrix mineralization, and these negative effects were reversed by PIK3R1 overexpression\u0026nbsp;(Fig. 3J). These results suggest that PIK3R1 partially blocks the impact of miR-103a-3p on DPSCs. Since PIK3R1 is a key component of the PI3K/AKT signaling pathway, which plays a crucial role in the odontogenic differentiation of DPSCs\u003csup\u003e14\u0026ndash;16\u003c/sup\u003e, we investigated whether PIK3R1 exerts its effects through this pathway. Overexpression of PIK3R1 in DPSCs significantly increased the p-AKT/AKT ratio, as shown by Western blot analysis (Fig. 3J). To verify PI3K/AKT pathway involvement, cells were then treated with the pathway inhibitor LY294002, which reduced p-AKT levels and partially reversed PIK3R1-induced odontogenic differentiation (Fig. 3K). These results suggest that PIK3R1 alleviates miR-103a-3p\u0026ndash;induced suppression of odontogenic differentiation, at least in part, through activation of the PI3K/AKT signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. KLF4 partially reverses miR-103a-3p\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003emediated suppression in DPSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also explored the role of KLF4 in odontogenic differentiation of DPSCs. Three KLF4-specific siRNAs (si-KLF4-1, si-KLF4-2, and si-KLF4-3) were synthesized and transfected into DPSCs, with si-KLF4-1, the most efficient knockdown, selected for further experiments (Fig. 4A). After 14 days of mineralization induction, odontogenic gene expression, ALP activity, and mineralized nodule formation were significantly reduced in the KLF4 knockdown group (Fig. 4B-D). Conversely, overexpression of KLF4 did the opposite, indicating its promotive role in odontogenic differentiation (Fig. 4E\u0026ndash;G). To determine whether KLF4 could counteract the inhibitory effects of miR-103a-3p, we conducted rescue experiments. miR-103a-3p mimics markedly suppressed odontogenic differentiation, whereas co-transfection with a KLF4 overexpression plasmid largely reversed these inhibitory effects (Fig. 4H\u0026ndash;J), suggesting that KLF4 partially blocks the negative impact of miR-103a-3p on DPSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. H19 promotes the odontogenic differentiation of DPSCs in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDPSCs with stable H19 overexpression (LV-H19) or control (LV-NC) cells were seeded into tooth root slices and implanted subcutaneously into BALB/c nude mice for 8 weeks (Fig. 5A-B). After implantation, pulp-like tissue formed within the root canal in the LV-H19 group (Fig. 5C). qRT-PCR analysis of regenerated tissues showed significantly higher H19 expression, elevated levels of odontogenic markers (ALP, RUNX2, DSPP, DMP1), and increased expression of target genes PIK3R1 and KLF4 compared to controls (Fig. 5D-G). H\u0026amp;E staining confirmed dentin-like tissue formation adjacent to the dentin matrix, while Masson\u0026rsquo;s trichrome staining revealed greater collagen deposition in the LV-H19 group (Fig. 5H-I). Immunofluorescence further demonstrated that PIK3R1 and KLF4 were predominantly localized within the newly formed dentin-like tissue (Fig. 5J). Collectively, these results indicate that H19 upregulates PIK3R1 and KLF4 expression and enhances the odontogenic differentiation of DPSCs in vivo.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDental pulp regeneration aims to reconstruct a functional pulp\u0026ndash;dentin complex by restoring pulp vitality and promoting dentin repair, ultimately enabling the recovery of tooth function\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Dental pulp stem cells (DPSCs) play a central role in this process, given their capacity to differentiate into odontoblast-like cells, repair hard dental tissues, and support pulp regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Understanding the molecular mechanisms governing DPSCs odontogenic differentiation is essential for optimizing regenerative endodontic strategies.\u003c/p\u003e\u003cp\u003eLong non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that participate in diverse cellular processes, including growth, pluripotency, and differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Our previous lncRNA-array analysis identified H19 as an upregulated transcript during odontogenic differentiation of DPSCs, and subsequent in vitro studies confirmed its promotive role\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To investigate its in vivo function, DPSCs overexpressing H19 (LV-H19) were implanted into tooth root segments and transplanted subcutaneously into nude mice. This treatment markedly enhanced dentin-like tissue formation and increased expression of odontogenic markers DSPP and DMP1, demonstrating that H19 promotes reparative dentinogenesis in vivo.\u003c/p\u003e\u003cp\u003eAlthough H19 has been implicated in the osteogenic and odontogenic differentiation of DPSCs, its precise molecular mechanism remains unclear. According to the competing endogenous RNA (ceRNA) hypothesis, lncRNAs can act as molecular sponges for microRNAs, thereby preventing them from suppressing their target mRNAs\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For example, H19 promotes MSC survival and angiogenic potential by sponging miR-199a-5p and upregulating its target gene VEGFA\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In this study, bioinformatics analysis predicted that miR-103a-3p could bind to lncRNA H19. While miR-103a-3p has been reported to inhibit osteogenic differentiation of ADSCs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, its role in DPSCs remains poorly understood. We therefore examined the interaction between miR-103a-3p and H19 and assessed its effect on odontogenic differentiation. Our results showed that H19 negatively regulates miR-103a-3p, and dual-luciferase assays confirmed their direct binding via a specific MRE, consistent with the ceRNA mechanism. Functionally, inhibition of miR-103a-3p enhanced odontogenic differentiation of DPSCs, as evidenced by increased expression of ALP, RUNX2, DSPP, and DMP1, together with greater matrix mineralization. In rescue experiments, miR-103a-3p mimics partially reversed the pro-odontogenic effects of H19, supporting the conclusion that H19 facilitates odontogenic differentiation by sponging miR-103a-3p through a ceRNA regulatory network.\u003c/p\u003e\u003cp\u003eThe PI3K/AKT signaling pathway plays a critical role in regulating cell proliferation, differentiation, and migration\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Its core components include phosphoinositide 3-kinase (PI3K), phosphatidylinositol-3,4,5-trisphosphate (PIP3), phosphoinositide-dependent kinase-1 (PDK1), and protein kinase B (AKT)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that this pathway is involved in the osteo/odontogenic differentiation of tooth-derived mesenchymal stem cells (MSCs)\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Bioinformatics analysis predicted that the 3\u0026prime; untranslated region (3\u0026prime;UTR) of PIK3R1 contains a sequence complementary to the seed region of miR-103a-3p, suggesting direct targeting. Interestingly, PIK3R1 encodes p85α, a regulatory subunit of the PI3K complex. This subunit activates the PI3K/AKT pathway by stabilizing and recruiting the catalytic subunit p110 to receptor tyrosine kinases, thereby promoting PIP3 production and subsequent AKT phosphorylation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We hypothesized that lncRNA H19 promotes odontogenic differentiation of DPSCs by sponging miR-103a-3p, thereby upregulating PIK3R1 and activating PI3K/AKT signaling. To test this, a dual-luciferase reporter assay confirmed direct binding between miR-103a-3p and PIK3R1. Consistently, miR-103a-3p mimics reduced PIK3R1 expression, whereas inhibitors increased it, indicating post-transcriptional suppression. We next assessed PIK3R1 function and found that its overexpression promoted odontogenic differentiation of DPSCs. In rescue experiments, co-transfection of PIK3R1 with miR-103a-3p mimics restored odontogenic gene expression compared with miR-103a-3p mimics alone. We further examined the relationship between PIK3R1 and PI3K/AKT signaling. As expected, PIK3R1 overexpression increased the p-AKT/AKT ratio, while treatment with an AKT pathway inhibitor reversed this effect and reduced odontogenic gene expression. Collectively, these findings demonstrate that H19 facilitates PIK3R1 expression by sponging miR-103a-3p, thereby enhancing PI3K/AKT signaling and promoting odontogenic differentiation in DPSCs.\u003c/p\u003e\u003cp\u003eKLF4 is a key transcription factor regulating stem cell pluripotency and differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR38 CR39 CR40 CR41\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that KLF4 binds to the promoters of mineralization-related genes, such as Dmp1 and Sp7, to regulate the odontogenic differentiation of DPSCs\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In KLF4 knockout mouse models, Chen et al. observed enlarged pulp chambers and defective dentin mineralization, underscoring its essential role in dentin formation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In this study, KLF4 was predicted and confirmed to be a direct target of miR-103a-3p. KLF4 overexpression promoted odontogenic differentiation, consistent with its reported positive role in odontogenesis. Rescue experiments further showed that KLF4 co-expression partially reversed the inhibitory effects of miR-103a-3p, indicating that H19 enhances odontogenic differentiation by upregulating KLF4 through miR-103a-3p sponging.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study highlights the odontogenic role of H19 in DPSCs, primarily through two key pathways: the lncRNA-H19/miR-103a-3p/PIK3R1/AKT axis and the lncRNA-H19/miR-103a-3p/KLF4 axis (Fig. 6). These findings provide new insights into the molecular mechanisms underlying dentin differentiation of DPSCs and may offer potential therapeutic targets for future regenerative strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ehDPSCs: Human dental pulp stem cells; lncRNA: Long non-coding RNA; ceRNA: Competing endogenous RNA; qRT-PCR: Quantitative real-time polymerase chain reaction; DSPP: Dentin sialophosphoprotein; DMP-1: Dental matrix protein-1; ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; PIK3R1: Phosphoinositide-3-Kinase Regulatory Subunit 1; KLF4: Kr\u0026uuml;ppel-like factor 4; PI3K: Phosphoinositide 3-kinase; AKT: AKT serine/threonine kinase; MSCs: Mesenchymal stem cells; miRNA: MicroRNA; 3\u0026prime;UTR: 3\u0026prime; untranslated region; NC: Negative control\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJKZ and LL contributed equally to this work and share first authorship. QZJ designed and conceived the study. JKZ critically performed the experiments. JKZ, LL, HXD, and GWC analyzed the data. JKZ, JLZ, CHW and BTW performed the statistical analysis. JKZ drafted the manuscript. JKZ, LL, QZJ, GWC and XSC critically revised the manuscript. QZJ provided funding. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Program of Guangdong Provincial Science and Technology Department (no. 2016ZC0134) and Guangdong Basic and Applied Basic Research Foundation (no. 2024A1515012741).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting this study are available in the article and its Supplementary Materials. The original sequencing data have been provided in the supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman dental pulp stem cells (hDPSCs) were purchased from Oricell (Shanghai, China). The original source (Oricell) has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. All animal experiments were reviewed, approved, and supervised by the Experimental Animal Ethics Committee of Ruiye Model Animal (Guangzhou) Biotechnology Co. Ltd (Approval No. RYEth-20241026574; Project title: Mechanism of long non-coding RNA H19 regulating dentin-oriented differentiation of human dental pulp stem cells; Date of approval: October 26, 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript has been approved by all authors. As this study did not involve human participants or individual human data, informed consent was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArtificial intelligence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not used AI‑generated work in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLin, L. \u003cem\u003eet al.\u003c/em\u003e circ_0002456/FUS interaction inhibits NF-\u0026kappa;B signaling to attenuate DNA damage and inflammatory responses in hDPSCs. \u003cem\u003eStem Cell Res. Ther.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 276 (2025).\u003c/li\u003e\n\u003cli\u003eCheng, L. \u003cem\u003eet al.\u003c/em\u003e Expert consensus on dental caries management. \u003cem\u003eInt. J. 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Oral Biol.\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 403\u0026ndash;411 (2009).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"H19, Odontogenic differentiation, Dental pulp stem cells","lastPublishedDoi":"10.21203/rs.3.rs-7499703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7499703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eFunctional regeneration of the dentin\u0026ndash;pulp complex is essential for restoring tooth integrity after injury. Odontoblastic differentiation of dental pulp stem cells (DPSCs) plays a central role in reparative dentinogenesis. Although lncRNA H19 is known to regulate biomineralization, its downstream network remains unclear. This study identified miR-103a-3p as a novel downstream effector of H19 and investigated its regulatory network in DPSCs odontoblastic differentiation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eInteractions between H19 and miR-103a-3p as well as miR-103a-3p and targets mRNAs (PIK3R1 and KLF4), were validated via bioinformatic and dual luciferase reporter assays. Quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of H19 and its potential signal axis and odontogenic markers. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to evaluated odontogenic ability. Finally, a heterotopic pulp regeneration model was established to reveal the regulating effects of H19.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eH19 acted as a sponge for miR-103a-3p, which otherwise inhibited the expression of odontogenic markers. PIK3R1 and KLF4 were identified as direct targets of miR-103a-3p. Overexpression of either PIK3R1 or KLF4 rescued the odontogenic differentiation capacity of DPSCs suppressed by miR-103a-3p. Furthermore, PIK3R1 promoted odontogenesis by activating the PI3K/AKT signaling pathway, while KLF4 functioned as an independent transcriptional regulator. Finally, in a heterotopic pulp regeneration model, H19 overexpression enhanced the expression of PIK3R1 and KLF4 and promoted odontoblastic differentiation of DPSCs in vivo.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur results suggest that the H19-mediated miR-103a-3p/PIK3R1/AKT and miR-103a-3p/KLF4 axes promote the odontogenic differentiation of DPSCs and are expected to serve as therapeutic targets for pulp regeneration.\u003c/p\u003e","manuscriptTitle":"H19 promotes odontogenic differentiation of human dental pulp cells via miR-103a-3p-mediated PIK3R1/AKT and KLF4 pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 16:37:19","doi":"10.21203/rs.3.rs-7499703/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d641686e-2c3d-4efa-8794-f991c59327ba","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-05T08:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 16:37:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7499703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7499703","identity":"rs-7499703","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}