USP7通过增强Notch1蛋白稳定性促进结肠癌增殖、迁移过程OA

Objective:To investigate the specific mechanisms by which the USP7/Notch1/Hes-1 signaling axis regulates the proliferation and migration of colorectal cancer(CRC)cells.Methods:The expression of USP7 and Notch1 in CRC was assessed using the ENCORI database(https://rnasysu.com/encori).The correlation between USP7 and Notch1 expression was analyzed utilizing the GEPIA database(http://gepia.cancer-pku.cn/).Protein expression levels of USP7 and Notch1 in CRC tissues and paired normal colon tissues were examined via The Human Protein Atlas(HPA)database.The protein-protein interaction net-work between USP7 and Notch1 was constructed using the STRING database.Molecular docking of USP7 and Notch1 was per-formed using HDOCK.The physical interaction between USP7 and Notch1 proteins was experimentally validated by co-immuno-precipitation(Co-IP).An USP7-overexpressing cell line was established.Western blot was employed to assess the protein expres-sion levels of Notch1 and its downstream effector,Hes-1.The effect of USP7 overexpression on Notch1 ubiquitination was further confirmed by immunoprecipitation(IP)analysis.To investigate the functional consequences,cell proliferation was evaluated using CCK-8 and colony formation assays following USP7 overexpression and concomitant Notch1 knockdown.Protein levels of Notch1 and Hes-1 under these conditions were again determined by Western blot.Cell migration capacity was assessed by wound healing(scratch)and Transwell assays.Additionally,Western blot was used to detect changes in the expression of epithelial-mesenchy-mal transition(EMT)markers,Vimentin and E-cadherin.Results:Compared to normal tissues(n=41),both USP7 and Notch1 were significantly upregulated in CRC tissues(n=471,P＜0.01).Consistent with this,protein expression levels of USP7 and Notch1 were also markedly higher in CRC tissues than in normal colon tissues.Furthermore,a significant correlation and di-rect interaction were identified between USP7 and Notch1.Overexpression of USP7 led to a significant increase in Notch1 and Hes-1 protein levels and a concomitant decrease in Notch1 ubiquitination.Functionally,USP7 overexpression significantly enhanced CRC cell clonogenicity and migration capacity(P＜0.05).Importantly,these pro-tumorigenic effects were reversed by concomi-tant Notch1 knockdown under USP7 overexpression conditions,resulting in significantly reduced colony formation and migration(P＜0.05).Conclusion:The deubiquitinating enzyme USP7 is highly expressed in CRC and promotes cancer cell proliferation and migration by stabilizing the Notch1 protein.