Interaction Between Deubiquitinating Enzyme OTUD5 And CacyBP And Bioinformatics Analysis Of OTUD5 In Cervical Cancer

Objective The study found that OTUD5(OTU deubiquitinase 5),as one of the important members of the OTU subfamily,plays an important role in the field of DNA damage repair and immunology.At present,OTUD5 research is relatively small,but whether it can be regulated still unknown.Calcyclin binding protein(CacyBP)is found as the target molecule of S100A6 protein in mouse Ehrlich ascites cell tumor.The study found that CacyBP affects the occurrence and development of a variety of tumors.In addition,there have been reports that overexpression of CacyBP in human glioma cells down-regulates p53 protein levels.At present,the function of OTUD5 in tumors is unclear,and the expression of OTUD5 in cervical cancer is analyzed by bioinformatics to provide reference for relevant research.Whether the deubiquitinating enzyme OTUD5 interacts with CacyBP and regulates cervical cancer is unclear.Methods Western blot experiments were used to detect the changes of foreign substrate protein after OTUD5 or CacyBP overexpression.RNA interference technology was used to knock down OTUD5 or CacyBP to detect changes in endogenous substrate proteins.Real-time quantitative PCR(qRT-PCR)was used to detect the effect of OTUD5 or CacyBP overexpression on substrate mRNA levels.The half-life test was used to detect the degradation rate of substrate protein OTUD5 after overexpression of CacyBP,and to explore the regulation of CacyBP on the stability of OTUD5.In vivo ubiquitination experiments were used to detect the effect of OTUD5 or CacyBP overexpression or knockdown on the level of substrate protein ubiquitination.Through nucleoplasm separation kit and Western blot,detect whether OTUD5 overexpression affects the cytoplasmic localization of CacyBP.Using Oncomine,GEPIA,UALCAN database to analyze the changes of OTUD5 mRNA level in CESC tissue.LinkedOmics was used to analyze the co-expressed genes and miRNA of the OTUD5 gene.The cBioPortal database was used to analyze the changes in DNA copy number of OTUD5 in CESC tissues.The GeneMANIA database was used to analyze the interacting proteins of OTUD5.The t test was used to verify the difference in OTUD5 mRNA expression between CESC and adjacent tissues.Spearman correlation coefficient was used to analyze the correlation of gene expression.Kaplan-Meier survival analysis was used to calculate survival rate,and generalized log-rank test was used to estimate the difference in survival rate.Results(1)CacyBP and OTUD5 have an interaction.Overexpression of CacyBP reduces the level of OTUD5 protein and shortens the half-life of OTUD5 protein,but has no effect on its m RNA level.CacyBP increases the ubiquitination level of OTUD5 protein.And CacyBP reduces the protein level of p53 through OTUD5.(2)OTUD5 does not affect the changes of CacyBP mRNA and protein levels,but the overexpression of OTUD5 promotes the nuclear localization of CacyBP.(3)The low expression of OTUD5 in cervical cancer is associated with poor prognosis.Its expression is related to the stage of cervical cancer and metastatic lymph nodes.In addition,the low expression of OTUD5 is related to different subtypes of cervical cancer,such as phosphatidylinositol-3-kinase(PI3K)-AKT signaling,epithelial-mesenchymal transition(EMT)and hormone-related subtypes.By analyzing the co-expressed genes of OTUD5,related miRNAs,transcription factors,kinases,E3 and interacting proteins.We demonstrated that OTUD5 affects the expression levels of WDR45,USP11,GRIPAP1 and RBM10.In addition,hsa-mir-137,hsa-mir-1913,hsa-mir-937,hsa-mir-607,hsa-mir-3149 and hsa-mir-144 may inhibit the expression of OTUD5.In summary,we conducted an enrichment analysis of 22 co-expressed genes,33 related miRNAs,and 30 interacting proteins.In addition to ubiquitination and immunology-related processes,they are also involved in Hippo signaling,insulin signaling,EMT,histone methylation,and phosphorylation kinase binding.Conclusion:By interacting with OTUD 5,CacyBP causes ubiquitination and degradation of OTUD5 protein and reduces its stability;OTUD5 promotes the nuclear localization of CacyBP and reduces its ubiquitination level.The study analyzed the expression of OTUD5 in cervical cancer and its relationship with clinicopathology for the first time,and provided new insights for further research on its regulatory mechanism in tumors.