Study On The Mechanism Of OTUD4 Deubiquitination And Modification Of B7-H4 To Regulate The Immune Microenvironment Of Breast Cancer

Objective: To elucidate the mechanism of OTUD4 deubiquitination to modify B7-H4,and the effect of this mechanism on the tumor microenvironment of breast cancer.Methods: In this project,the expression of B7-H4 in breast cancer cells was located by immunohistochemical method,and the correlation between the expression level of B7H4 and the prognosis of breast cancer patients was calculated;Western Blot and RT-q PCR were used to detect the expression of B7-H4 in common breast cancer cells Whether the expression of protein level and m RNA level in the line is consistent.The ubiquitination modifying enzyme OTUD4 interacting with B7-H4 was screened out by protein co-immunoprecipitation-mass spectrometry.Exogenous and endogenous Co-IP experiments explored whether there was interaction with OTUD4 and B7-H4.Breast cancer cell lines with OTUD4 overexpression and OTUD4 knockdown were stably constructed with lentivirus,and the changes of B7-H4 protein and m RNA expression levels in breast cancer cell lines after OTUD4 overexpression and OTUD4 knockdown were detected by Western Blot and RT-q PCR.The protein immunoprecipitation-ubiquitination experiment further explored the effect of OTUD4 on the ubiquitination modification of B7-H4,and the protein half-life experiment explored the regulation of OTUD4 overexpression and OTUD4 low expression on the half-life of B7-H4 protein.Clonogenic experiments,cell proliferation experiments and Transwell experiments proved the effects of OTUD4 and B7-H4 on the growth,proliferation and metastasis of breast cancer cells.The relationship between the expression level of B7-H4 and the infiltration of CD8+ T cells in the tumor microenvironment was analyzed by multicolor fluorescent immunoassay.Results: The expression of B7-H4 was up-regulated in breast cancer,and the expression level was related to the postoperative recurrence and metastasis of patients;the protein level and RNA level expression of B7-H4 in breast cancer cell lines were inconsistent,and there was obvious ubiquitination in breast cancer cell lines Modification regulation;OTUD4 interacts with B7-H4 and positively regulates B7-H4 at the protein level;OTUD4 promotes the stability of B7-H4 protein by deubiquitinating B7-H4 molecules;OTUD4 and B7-H4 do not affect breast cancer cells The level of growth,proliferation and migration itself;OTUD4 affects the tumor immune microenvironment and the infiltration of CD8+ T cells by stabilizing the expression of B7-H4.Conclusion: The expression of B7-H4 in breast cancer tissue is higher than that in normal tissue,and the prognosis of breast cancer patients with high expression of B7-H4 is worse.OTUD4 can positively regulate B7-H4 expression through deubiquitination.The expression of OTUD4 and B7-H4 affects the metastasis and prognosis of breast cancer by affecting the infiltration of CD8+ T cells.