USP4 Mediates The Role Of SOX9 In Regulating Wnt/β-Catenin Signaling Pathway In Colorectal Cancer

Object: This study mainly discusses the effects of USP4 on the invasion and stemness of colorectal cancer.To further clarify the regulatory effect and mechanism of USP4 on SOX9,and provide a new target for the treatment of colorectal cancer in the future.Methods: 1.Definition of USP4 and SOX9 expression in normal colon cells and colorectal cancer cell lines by using the Western blot method.2.The proliferation,migration and invasion ability of colorectal cancer cells were determined by respectively using MTT and Transwell tests.And the expressions of EMT and stemness proteins were detected by Western blot.3.Ubi Browser website was used to analyze and screen the substrate proteins of USP4.USP4 was up-regulated or down-regulated in SW480 and HCT116 cells,and the changes in SOX9 m RNA and protein levels were detected by q RT-PCR and Western blot.The interaction of USP4 and SOX9 was determined by co-immunoprecipitataion and Western blot.4.USP4 was upregulated or down-regulated in SW480 cells,and protein synthesis inhibitor CHX was added to observe the degradation rate of SOX9 protein in USP4 up-regulated group and USP4 down-regulated group.In SW480 cells with USP4 knockdown,MG132 was added to prevent protein degradation,and the changes of SOX9 protein were observed compared with the control group and the USP4 down-regulated group.5.The deubiquitination experiment was conducted to detect whether SOX9 could be deubiquitinated by USP4.6.USP4 overexpression plasmid and SOX9-si RNA or USP4-si RNA and SOX9 overexpression plasmid were co-transfected into SW480 cells,western blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteins.Results: 1.The results of Western blot indicated that USP4 and SOX9 were significantly higher in colorectal cancer cells than in normal colon.2.MTT and Transwell assay indicated that USP4 could promote the proliferation,migration and invasion of colorectal cancer cells.Western blot results showed that the expression of EMT and stemness related proteins decreased after USP4 knockdown.3.Ubi Browser analysis and transcriptome sequencing results confirmed that SOX9 was the new substrate protein.q RT-PCR and Western blot analysis showed that USP4 up-regulated SOX9 protein expression,but did not affect SOX9 m RNA level.Through the immune coprecipitation experiment,confirmed the interaction between USP4 and SOX9.4.USP4 could prolong the protein half-life of SOX9.5.Deubiquitination assay showed that SOX9 was deubiquitinated by USP4.6.Western blot showed that USP4 could increase the expression of Wnt/β-catenin signaling pathway-related proteins.Moreover,the results of reversion assay indicated that USP4-mediated SOX9 regulated Wnt/β-catenin signaling pathway.Conclusions: 1.USP4 promotes the proliferation,migration and invasion of colorectal cancer cells.2.USP4 promotes the stemness and EMT of colorectal cancer cells.3.USP4 interacts with SOX9,and USP4 maintains the stability of SOX9 protein through deubiquitination.4.USP4 mediates SOX9 to regulate the Wnt/β-catenin pathway.