[摘 要] 目的：探讨DNA甲基转移酶1（DNMT1）通过稳定zeste基因增强子同源物2（EZH2）促进结直肠癌（CRC）HCT8细胞 增殖与迁移的机制。方法：利用生物信息学方法分析 DNMT1 在 CRC 组织中的表达水平。WB 法检测 DNMT1 在 CRC 细胞 HCT8、SW620和正常结肠上皮细胞NCM460中的表达。通过siRNA或慢病毒载体转染HCT8细胞，分为siNC组、siDNMT1组、 Vector组、DNMT1-OE组、siTRAF6组、siEZH2组、siEZH2 + DNMT1-OE组。采用克隆形成实验、CCK-8法、Transwell实验和划痕 愈合实验检测敲低或过表达DNMT1对HCT8细胞增殖与迁移的影响，WB和qPCR法检测EZH2蛋白和mRNA水平，免疫沉淀 （IP）法检测EZH2泛素化水平，免疫荧光双染检测肿瘤坏死因子受体相关因子6（TRAF6）与EZH2的细胞内共定位情况，克隆形 成和划痕愈合实验验证EZH2对DNMT1功能的逆转作用。收集2022—2025年间郑州大学附属洛阳中心医院手术切除的12例 CRC患者的癌及癌旁组织标本，采用免疫组化法检测CRC组织中DNMT1、TRAF6和EZH2的表达水平。结果：DNMT1在CRC 组织中表达显著高于癌旁组织（P < 0.01），且在CRC细胞中表达上调（P < 0.05）；DNMT1 敲低显著抑制 HCT8 细胞增殖及迁 移（均 P < 0.01），过表达则相反（均 P < 0.01）。DNMT1 正向调控 EZH2 的蛋白水平（P < 0.01），而 mRNA 水平不变（P > 0.05）。 MG132可恢复siDNMT1组的EZH2蛋白表达（P < 0.01），且 siDNMT1 组 EZH2 泛素化水平升高。DNMT1 负向调控 TRAF6 的表达（P < 0.01），且TRAF6与EZH2在细胞质中共定位，IP证实两者直接结合。敲低TRAF6可减弱EZH2的泛素化水平，敲低 EZH2可逆转DNMT1对HCT8细胞增殖、迁移的促进作用（均P < 0.01）。DNMT1和EZH2在CRC组织中呈高表达（P < 0.01）， TRAF6在CRC组织中表达显著低于癌旁组织（P < 0.05）。结论：DNMT1通过抑制TRAF6稳定EZH2促进CRC细胞的增殖和迁 移，DNMT1、TRAF6和EZH2可能是CRC治疗的潜在靶点。

[Abstract] Objective: To explore the mechanisms by which DNA methyltransferase 1 (DNMT1) promotes the proliferation and migration of colorectal cancer (CRC) HCT8 cells through the stabilization of enhancer of zeste homolog 2 (EZH2). Methods: Bioinformatic analysis was performed to evaluate the expression level of DNMT1 in CRC tissues. Western blotting (WB) analysis was used to determine DNMT1 expression levels in CRC cell lines HCT8 and SW620, as well as in normal colon epithelial cells NCM460. HCT8 cells, after transfection with siRNA or lentivirus, were assigned to the following groups: the siNC group, the siDNMT1 group, the Vector group, the DNMT1-OE group, the siTRAF6 group, the siEZH2 group, the siEZH2 + DNMT1-OE group. The effects of DNMT1 knockdown or overexpression on the proliferation and migration of HCT8 cells were assessed by colony formation assay, CCK-8 assay, Transwell assay, and scratch assay. The protein and mRNA levels of EZH2 were measured by WB and quantitative realtime PCR (qPCR), respectively. EZH2 ubiquitination levels were examined using immunoprecipitation (IP). Intracellular colocalization of TRAF6 and EZH2 was evaluated by immunofluorescence double staining. The reversing effects of EZH2 on DNMT1 was confirmed by colony formation and wound healing (scratch) assays. Cancerous tissue and adjacent tissue specimens of 12 CRC patients surgically removed at Luoyang Central Hospital affiliated to Zhengzhou University between 2022 and 2025 were collected and immunohistochemical staining was performed to detect the expression levels of DNMT1, TRAF6, and EZH2 in CRC tissue samples. Results: DNMT1 expression was significantly higher in CRC tissues than in adjacent non-tumor tissues (P < 0.01), and its expression was also upregulated in CRC cells (P < 0.05). Knockdown of DNMT1 markedly inhibited the proliferation (P < 0.01) and migration (P < 0.01) of HCT8 cells, whereas overexpression of DNMT1 produced the opposite effects (both P < 0.01). DNMT1 positively regulated EZH2 at the protein level (P < 0.01) but did not affect its mRNA expression (P > 0.05). MG132 restored the protein expression of EZH2 (P < 0.01) and increased EZH2 ubiquitination levels in the siDNMT1 group, DNMT1 negatively regulated TRAF6 expression (P < 0.01). TRAF6 and EZH2 co-localized in cytoplasm, and IP confirmed that they bound directly. Knockdown of TRAF6 reduced EZH2 ubiquitination levels. EZH2 knockdown reversed the promotive effects of DNMT1 on HCT8 cell proliferation (P < 0.01) and migration (P < 0.01). DNMT1 and EZH2 were highly expressed in CRC tissues (P < 0.01), whereas TRAF6 expression was significantly lower in CRC tissues than in adjacent non-tumor tissues (P < 0.05). Conclusion: DNMT1 promotes the proliferation and migration of CRC cells through suppressing the stabilization of EZH2 by TRAF6. DNMT1, TRAF6, and EZH2 are expected to become diagnostic markers and therapeutic targets for CRC.

[基金项目] 2025年河南省医学科技攻关联合共建项目（LHGJ20250785）；2023年河南省医学科技攻关计划项目 （LHGJ20230815）