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DNMT1 promotes the proliferation and migration of colorectal cancer HCT8 cells by suppressing TRAF6-mediated ubiquitination of EZH2
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Abstract:
[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.
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