Journal of Capital Medical University ›› 2025, Vol. 46 ›› Issue (6): 992-999.doi: 10.3969/j.issn.1006-7795.2025.06.006

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Micro RNA-10b regulates the early tooth development homeostasis of miniature swine by targeting and suppressing Wnt9B

Sun Meng1, He Xiaoli1, Tong Xiangyao1, Li Ang1, 2*   

  1. 1.Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an 710049, China; 2.Department of Periodontology, College of Stomatology, Xi'an Jiaotong University, Xi'an 710049, China
  • Received:2025-09-22 Revised:2025-10-10 Online:2025-12-21 Published:2025-12-19
  • Supported by:
    The studty was supported by  National Natural Science Foundation of China (82370939).

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Abstract: Objective  To investigate of the regulatory role of micro RNA (miR)-10b in early tooth development homeostasis of miniature swine and its underlying molecular mechanisms.Methods  Miniature swine bud-stage first deciduous molar tooth germs were used to perform miRNA sequencing under monoculture and mandible co-culture conditions to identify differentially expressed miRNAs. Among these, miR-10b was selected for further study, and its expression was validated by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Dental mesenchymal cells were transfected with lentiviral vectors (LV) in vitro to establish a miR-10b overexpression model, and cell proliferation was evaluated using EdU and CCK-8 assays. Potential target genes of miR-10b were predicted through TargetScan and miRDB databases, and the binding sites within the 3'-untranslated region (3'-UTR) were further analyzed with RNAhybrid 2.2. To verify direct interactions, wild-type (WT) and mutant (MUT) pmirGLO dual-luciferase reporter constructs were generated, followed by dual-luciferase reporter assays and RT-qPCR to confirm the regulatory relationship.Results  Sequencing analysis revealed that miR-10b was significantly upregulated in the mandible co-culture group compared with the tooth germ monoculture group (log2FC=4.82, P<0.05), which was confirmed by RT-qPCR (2.03 ± 0.21 fold increase, P<0.001), suggesting its involvement in tooth development in miniature swine. To investigate the functional effects of miR-10b, it was overexpressed in dental mesenchymal cells, and further verified by RT-qPCR (P<0.01). EdU and CCK-8 assays showed that overexpression of miR-10b significantly inhibited the proliferation of dental mesenchymal cell. Bioinformatic analysis identified wingless-type MMTV integration site family, member 9B (Wnt9B) as a potential target gene of miR-10b and showed that specific binding sites were located within the 3'-UTR. Dual-luciferase reporter assays demonstrated direct interaction and showed that miR－10b mimics significantly decreased luciferase activity in Wnt9B-WT (P<0.01) but did not affect Wnt9B-MUT. Consequently, overexpression of miR-10b markedly downregulated Wnt9B mRNA levels in dental mesenchymal cells (P<0.01).Conclusion  miR-10b can decrease the proliferation activity of dental mesenchymal cells, regulate the homeostasis during early tooth development and maintain the balance of mandible-teeth communication by targeting and inhibiting the expression of Wnt9B in miniature swine. This study provides a new theoretical clue for revealing the molecular mechanisms of tooth development and regeneration strategies.

Key words: tooth development, miniature swine, cell proliferation, microR-10b, Wnt 9B, dental mesenchymal cells

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