Park Seung Gwa, Kim Ki-Tae, Kim Woo-Jin, Kim Sungtae, Cho Young-Dan
Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea.
Dental Multiomics Center, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea.
Epigenomics. 2025 Aug 12:1-11. doi: 10.1080/17501911.2025.2544513.
In elderly patients, bone regeneration is impeded by age-related shifts in mesenchymal stem cell differentiation propensity toward adipogenesis over osteogenesis. We investigated whether DNA demethylation by 5‑aza‑2'‑deoxycytidine (5azaC) synergizes with Wnt Family Member 3A (Wnt3a) signaling to induce osteogenic potential in 3T3‑L1 pre-adipocytes, generating osteoblast-like cells.
3T3‑L1 pre-adipocytes were treated with 5azaC and/or Wnt3a. Osteogenic differentiation was assessed via ALP activity, mineralization assays, and marker expression. Transcriptomic and epigenomic profiling were performed and compared with MC3T3-E1 cells. Functional relevance of candidate genes was examined using siRNA knockdown.
Transcriptomic and epigenomic profiling revealed that 5azaC and Wnt3a co-treatment induced broader gene expression and methylation changes than either treatment alone, closely resembling the osteogenic profile of MC3T3-E1 pre-osteoblasts. Among the overlapping differentially methylated and steadily expressed genes, Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Eta ( and Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Epsilon ( emerged as key regulators, whose knockdown notably enhanced Alpl expression even without 5azaC.
Combining 5azaC-induced demethylation with Wnt3a is a potent strategy to redirect pre-adipocytes toward osteogenesis. Identification of key targets like Ywhah and Ywhae provides mechanistic insight into trans-differentiation and suggests therapeutic potential for bone regeneration, particularly in elderly periodontal patients.
在老年患者中，间充质干细胞分化倾向于向脂肪生成而非骨生成转变，这阻碍了骨再生。我们研究了5-氮杂-2'-脱氧胞苷（5azaC）诱导的DNA去甲基化是否与Wnt家族成员3A（Wnt3a）信号协同作用，以诱导3T3-L1前脂肪细胞的成骨潜能，从而生成类成骨细胞。
用5azaC和/或Wnt3a处理3T3-L1前脂肪细胞。通过碱性磷酸酶（ALP）活性、矿化分析和标志物表达评估成骨分化。进行转录组和表观基因组分析，并与MC3T3-E1细胞进行比较。使用小干扰RNA（siRNA）敲低技术检测候选基因的功能相关性。
转录组和表观基因组分析显示，与单独使用任何一种处理相比，5azaC和Wnt3a联合处理诱导了更广泛的基因表达和甲基化变化，与MC3T3-E1前成骨细胞的成骨谱非常相似。在重叠的差异甲基化和稳定表达基因中，酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白η（Ywhah）和酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白ε（Ywhae）成为关键调节因子，敲低它们即使在没有5azaC的情况下也能显著增强Alpl的表达。
将5azaC诱导的去甲基化与Wnt3a相结合是一种将前脂肪细胞重定向为成骨的有效策略。鉴定Ywhah和Ywhae等关键靶点为转分化提供了机制性见解，并提示了骨再生的治疗潜力，特别是在老年牙周病患者中。
