Ding Yulin, Lu Genqi, Zhao Ya, Zhang Yi, Zhang Jing, Ma Jingle, Yuan Yunyun, Liu Boyu, Liu Wei, Shen Wenjing
Department of Prosthodontics, Hebei Key Laboratory of Stomatology/ Hebei Technology Innovation Center of Oral Health, School and Hospital of Stomatology, Hebei Medical University, Shijiazhuang, 050017, China.
Department of Stomatology, The No. 2 Hospital of Baoding, Baoding, 071051, China.
BMC Oral Health. 2025 Mar 8;25(1):358. doi: 10.1186/s12903-025-05741-9.
Variants of Ectodysplasin A1 (EDA1) regulate the proliferation, migration, and odontogenic differentiation of human dental pulp stem cells (hDPSCs). Further study of these variants could reveal the mechanism by which EDA1 induces tooth development.
The following groups of hDPSCs were studied: those expressing wild-type (Wt) EDA1, those expressing EDA1 non-syndromic tooth agenesis (NSTA) variants (NSTA-A259E, NSTA-S374R), those expressing a syndrome type (STA) variant of EDA1 (STA-H252L), and those transformed with the empty vector (NC, negative control). hDPSCs proliferation was assessed using Cell Counting kit 8 assays. Flow cytometry was employed to assess hDPSCs cell cycle distribution. Transwell and wound-healing assays were employed to assess hDPSCs migration. hDPSCs mineralization was induced using odontogenic differentiation medium. RNA sequencing of the various hDPSCs groups was carried out to identify enriched pathways and hub genes. Hub gene expression was confirmed using quantitative realtime reverse transcription PCR (qRT-PCR).
Wt-EDA1 promoted hDPSCs proliferation and G0/G1 to S transition significantly compared with the NSTA-EDA1 and STA-EDA1 groups (p < 0.01). The NSTA-EDA1 and STA-EDA1 groups did not show significant differences between them (p > 0.05). Relative to that in the NSTA-EDA1 and STA-EDA1 groups, Wt-EDA1 enhanced hDPSCs migration (p < 0.01). According to alkaline phosphatase and Alizarin Red staining, compared to the Wt-EDA1 group, hDPSCs odontogenic differentiation was inhibited and proliferation was ablated in the NSTA-EDA1 and STA-EDA1 groups (p < 0.01). RNA sequencing showed enrichment of the MAPK signaling and osteoclast differentiation pathways, identifying FOS and JUN as differentially expressed hub genes. qRT-PCR demonstrated that, unlike the Wt-EDA1 group, the EDA1 variant groups could not promote FOS mRNA expression.
In hDPSCs, EDA1 variants could not promote FOS expression, which inhibited hDPSCs odontogenic differentiation and ablated their proliferation.
外胚层发育不良蛋白A1（EDA1）的变体调节人牙髓干细胞（hDPSCs）的增殖、迁移和牙源性分化。对这些变体的进一步研究可能揭示EDA1诱导牙齿发育的机制。
研究了以下几组hDPSCs：表达野生型（Wt）EDA1的细胞、表达EDA1非综合征性牙齿发育不全（NSTA）变体（NSTA-A259E、NSTA-S374R）的细胞、表达EDA1综合征型（STA）变体（STA-H252L）的细胞，以及用空载体转化的细胞（NC，阴性对照）。使用细胞计数试剂盒8检测评估hDPSCs的增殖。采用流式细胞术评估hDPSCs的细胞周期分布。采用Transwell和伤口愈合检测评估hDPSCs的迁移。使用牙源性分化培养基诱导hDPSCs矿化。对不同组的hDPSCs进行RNA测序，以鉴定富集的通路和枢纽基因。使用定量实时逆转录PCR（qRT-PCR）确认枢纽基因的表达。
与NSTA-EDA1和STA-EDA1组相比，Wt-EDA1显著促进hDPSCs增殖以及G0/G1期向S期的转变（p < 0.01）。NSTA-EDA1组和STA-EDA1组之间未显示出显著差异（p > 0.05）。相对于NSTA-EDA1和STA-EDA1组，Wt-EDA1增强了hDPSCs的迁移能力（p < 0.01）。根据碱性磷酸酶和茜素红染色结果，与Wt-EDA1组相比，NSTA-EDA1和STA-EDA1组中hDPSCs的牙源性分化受到抑制，增殖被消除（p < 0.01）。RNA测序显示丝裂原活化蛋白激酶（MAPK）信号通路和破骨细胞分化通路富集，鉴定出FOS和JUN为差异表达的枢纽基因。qRT-PCR表明，与Wt-EDA1组不同，EDA1变体组不能促进FOS mRNA表达。
在hDPSCs中，EDA1变体不能促进FOS表达，这抑制了hDPSCs的牙源性分化并消除了它们的增殖。
