The Mechanism Of USP39 Activating USP4 To Enhance The Activity Of TGF-β Signal Pathway

Objective: To analyze the relationship between USP4 and transforming growth factor-β type I receptor(TGFβRI)protein.To study the deubiquitination regμlation of ubiquitinspecific protease USP4 on TGFβRI.To detect the changes in EMT marker molecμles after activation of TGF-β signaling pathway.To study the relationship between USP4 expression and the regμlation on biological activity of breast cancer cells.To study the relationship between USP39 expression and the regμlation on biological activity of breast cancer cells.Methods: To analyze the relationship between USP4 and TGFβRI with coimmunoprecipitation experiment.To construct the stable breast cancer cell line BT-549 with USP4 over-expression,USP4 silencing by recombinant lentivirus infection.Throμgh specific ubiquitination experiments to examine the effect of USP4 on the ubiquitination level of TGFβRI.Using Western blot to detect the expression of EMT marker molecμles in breast cancer cells after the TGF-β signaling pathway was activated by TGFβ1.The effects of overexpression of USP4 on breast cancer cell migration,clonogenicity and proliferation were studied by wound healing experiment,cell clone formation experiments and CCK-8 experiments.The effects of overexpression of USP39 based on overexpression of USP4 on breast cancer cell migration,clonogenicity and proliferation were studied by wound healing experiment,cell clone formation experiments and CCK-8 experiments.Results: USP4 and TGFβRI can interact with each other in breast cancer cell line BT-549.The relative level of USP4 mRNA and protein in the USP4 over-expression stable breast cancer cell lines were 14.22 and 4.99 times respectively compared with the control cells.The relative level of USP4 mRNA in the USP4 silencing stable breast cancer cell lines was eighty-two percent of the control cells’.And the expression of USP4 protein was also significantly lower than the control cells.The ubiquitination level of TGFβRI was significantly decreased with USP4 overexpression and the ubiquitination level of TGFβRI was significantly increased with USP4 silencing.The morphological changes of EMT were observed in the two breast cancer cells after induced by TGFβ1,and trend of BT-549 cells with USP4 over-expression was more obvious.After induction,the expressions of N-cadherin,β-catenin,Vimentin and Slug were up-regμlated and the expression of ZO-1 protein was down-regμlated.The migration,clonogenicity and proliferation were showed significantly enhanced in the cells with USP4 over-expression.The migration,clonogenicity and proliferation were showed significantly further enhanced in the cells with overexpression of USP39 based on overexpression of USP4.Conclusions: TGFβRI and USP4 can interact in vivo.The ubiquitination level of TGFβRI was significantly decreased with USP4 over-expression and the ubiquitination level of TGFβRI was significantly increased with USP4 silencing.TGFβ1can induce the activation of TGF-β signaling pathway in BT-549 cells.Overexpression of USP4 can further enhance the activity of TGF-β signaling pathway and promote EMT in breast cancer cells.Overexpression of USP4 can enhance the biological activity of breast cancer cells.Overexpression of USP39 based on overexpression of USP4 can further enhance the biological activity of breast cancer cells.