首都医科大学学报 ›› 2025, Vol. 46 ›› Issue (6): 992-999.doi: 10.3969/j.issn.1006-7795.2025.06.006

• 稳态医学口腔专题 • 上一篇    下一篇

微小RNA-10b靶向抑制Wnt9B调控小型猪牙齿早期发育稳态的机制研究

孙萌1，贺晓利1，仝相瑶1，李昂1，2*   

  1. 1.西安交通大学口腔医院，陕西省颅颌面重点实验室，西安  710049; 2.西安交通大学口腔医院牙周科，西安 710049
  • 收稿日期:2025-09-22 修回日期:2025-10-10 出版日期:2025-12-21 发布日期:2025-12-19
  • 通讯作者: 李昂 E-mail:drliang@mailxjtu.edu.cn
  • 基金资助:
    国家自然科学基金项目（82370939）。

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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摘要： 目的  探索微小RNA（microRNA, miR）－10b 在小型猪牙齿早期发育稳态中的调控作用及潜在分子机制。方法  收集单独培养和与颌骨共培养的蕾状期小型猪乳磨牙胚，进行miRNA测序分析并筛选出差异表达 miR-10b，实时反转录定量聚合酶链反应（real-time reverse transcription quantitative polymerase chain reaction, RT-qPCR）验证其表达差异。体外利用慢病毒载体(lentivirus vector, LV）转染牙胚间充质细胞，构建 miR-10b 过表达模型，采用 EdU 标记法和细胞计数试剂盒－8（cell counting kit-8, CCK-8） 检测细胞增殖能力。TargetScan、miRDB 等数据库预测 miR-10b 潜在靶基因，并结合 RNAhybrid 2.2 软件分析靶基因 3'－非翻译区（3'-untranslated region 3'-UTR）结合位点。构建靶基因野生型（wild type, WT）及突变型（mutant, MUT）双萤光素酶 pmirGLO 报告载体，双萤光素酶报告实验及 RT-qPCR 验证 miR-10b 与靶基因间的靶向调控关系。结果  测序结果显示，与牙胚单独培养组（对照组）相比，牙胚与下颌骨共培养组（实验组）中 miR-10b 显著上调（log2FC=4.82，P<0.05）。RT-qPCR 验证结果与测序数据一致，实验组 miR-10b 相对表达量为对照组的 2.03±0.21 倍（P<0.001）。EdU 标记法和 CCK-8 实验结果均表明，miR-10b 的过表达显著抑制了间充质细胞的增殖能力（P<0.01）。生物信息学预测显示 Wnt家族成员9B (wingless-type MMTV integration site family, member 9B, Wnt9B) 为 miR-10b 的潜在靶基因，且两者 3'-UTR 存在特异性结合位点。双萤光素酶实验表明，与 Wnt9B-WT+mimics NC 组相比，Wnt9B-WT+miR-10b mimics 组萤光素酶活性显著降低（P<0.01），而 Wnt9B-MUT 组差异无统计学意义（P >0.05）；RT-qPCR 结果显示，miR－10b 过表达可显著下调牙胚间充质细胞中 Wnt9B 的 mRNA 表达水平（P<0.01）。结论  miR-10b 靶向抑制 Wnt9B 的表达下调牙胚间充质细胞增殖活性，进而参与小型猪牙齿早期发育过程中的稳态调控，维持颌骨－牙齿的通讯平衡，揭示牙齿发育分子机制并为再生策略提供新的理论依据。

关键词: 牙齿发育, 小型猪, 细胞增殖, 微小RNA －10b, Wnt家族成员9B, 牙胚间充质细胞

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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