Study Of ARRDC1 And ARRDC3 On Biological Behavior And Mechanism Of Renal Cell Carcinoma By Regulating YAP1

Object:Yes-associated protein 1(YAP1),one key downstream transcription co-activator of Hippo pathway and containing two WW domains,has been shown to be essential for cancer initiation,progression,or metastasis in clear cell renal cell carcinoma(cc RCC).The ?-arrestins domain-containing 1 and 3(ARRDC1 and ARRDC3),containing two PPXY motifs,are two members of ?-arrestins family.It has been reported that the WW domains can interact with the PPXY motifs.However,there is no specific study on the role and mechanism of these proteins in the tumorigenesis of renal cell carcinoma.Therefore,the aim of this work was to investigate the interaction between YAP1 and ARRDC1/3 in order to elucidate the relationship between ARRDC1 and ARRDC3 downregulation and cc RCC tumorigenesis.Methods:1.The expressions of ARRDC1 and ARRDC3 proteins in clear cell renal cell carcinoma and adjacent normal tissues were detected by Western blotting.2.Immunohistochemical method was used to detect the expression of ARRDC3 protein in clear cell renal cell carcinoma and adjacent normal tissues,and its correlation analysis was made with clinical pathological factors.3.Low ARRDC1 and ARRDC3 expressed 786-O cell lines of human clear cell renal cell carcinoma line were constructed and used to detect the cell proliferation,invasion and metastasis.4.YAP1 was identified as potential ARRDC1 and ARRDC3 interacting protein by tandem affinity purification/mass spectrometry.5.The interaction between YAP1 and ARRDC1/3 was identified by immunoprecipitation assay,and the mutual interaction domain was identified.Regulation of YAP1 ubiquitination was studied by ubiquitination assay.Results:1.ARRDC1 and ARRDC3 were pooly expressed in clear cell renal cell carcinoma.2.The expression of ARRDC3 was correlated with clinicopathological factors of clear cell renal cell carcinoma.3.ARRDC1 and ARRDC3 has the inhibitory effect of renal cell carcinoma,and its down-expression can promote the proliferation,metastasis and invasion of clear cell renal cell carcinoma by regulating YAP1.4.YAP1 is the interaction protein of ARRDC1 and ARRDC3,and both of them interact with each other through a specific domain.5.ARRDC1 and ARRDC3 reduce YAP1 stability by ubiquitination of YAP1.Conclusions:In this study,we conducted a great many experiments to reveal a novel mechanism for ARRDC1/3 involved in YAP1 stability regulation,and provid insight in understanding the relationship between ARRDC1/3 downregulation and aberrant Hippo-YAP1 pathway activation in cc RCC.