Study On The Cooperation Of TMEPAI And Nedd4 In Regulating NF-κB Signaling Pathway

NF-κB signaling is abnormally activated in a variety of cancers and is involved in tumor development by regulating the expression of genes related to cell proliferation,invasion,and inflammatory responses.Transmembrane prostate androgen-induced protein(TMEPAI)is highly expressed in various tumor tissues and is closely related to the occurrence and development of tumors.TMEPAI could interact with IκBα in vitro was found in our preliminary research,and another study reported that TMEPAI could interact with Nedd4.This paper will further analyze the mechanism by which TMEPAI promotes tumorigenesis and development and deeply study the interaction between TMEPAI,Nedd4 and IκBα,and the role and function of the three in synergistic regulation of NF-κB signaling.The results obtained in this paper include:TMEPAI,Nedd4 knockout He La cell lines were successfully constructed by CRISPRCas9 technique.Co-immunoprecipitations demonstrated that TMEPAI,Nedd4 and IκBαform a ternary complex by interaction in cells.TMEPAI acted as an adaptor protein to mediate the interaction between Nedd4 and IκBα in vivo.In vivo ubiquitination assay showed that knocking out TMEPAI,Nedd4 inhibited the ubiquitination of IκBα.Overexpression of TMEPAI and Nedd4 could synergistically enhance the ubiquitination of IκBα,while the expression of TMEPAI-2YA mutant plasmids could not,indicating that TMEPAI and Nedd4 synergistically promoted the ubiquitination and degradation of IκBα depends on the interaction between TMEPAI and Nedd4.Luciferase reporter assay,immunofluorescence and q RT-PCR assay were used to explore the synergistic activation of NF-κB signaling by TMEPAI and Nedd4.It was demonstrated that TMEPAI and Nedd4 could synergistically promote the activity of the NF-κB signaling reporter gene p NF-κB-Luc,trigger p65 nuclear translocation,and subsequent activation of expression of several downstream target genes of NF-κB including Cyclin D1,c-Myc,ICAM-1,MMP9,i NOS,COX-2 and TNF-α.MTT and Ki-67 experiments show that TMEPAI and Nedd4 can synergistically enhance He La cell viability and promote cell proliferation,and the growth-promoting activities of TMEPAI and Nedd4 depend on the activation of NF-κB signaling.Animal studies showed that knockout of TMEPAI and Nedd4 could inhibit the growth of tumor-bearing nude mice.The average tumor weight of the control group was 0.49 g,while the average tumor weight of the knockout TMEPAI and Nedd4 groups was only 0.07 g and 0.18 g and knockout of TMEPAI and Nedd4 in tumor tissue can also inhibit the degradation of IκBα.The research in this thesis shows that TMEPAI,Nedd4 and IκBα can form ternary complex in cells.TMEPAI degrades IκBα by recruiting Nedd4,promotes the entry of p65 into the nucleus,activates NF-κB signaling,and promotes the transcription of downstream target genes of NF-κB,thereby promoting the occurrence and development of tumors.This paper reveals a new mechanism by which TMEPAI promotes tumorigenesis and development by activating NF-κB signaling,and lays a theoretical foundation for treating tumors with TMEPAI as a target.