Function And Molecular Mechanism Of Deubiquitinating Enzyme OTUB2 In Intrahepatic Cholangiocarcinoma

BackgroundCholangiocarcinoma(CCA)is a malignant tumor originating from the intrahepatic,perihilar or distal(extrahepatic)biliary tract system.It is currently the second most common primary hepatic malignant tumor after hepatocellular carcinoma.Depending on the anatomical location,CCA including perihilor cholangiocarcinoma(p CCA),distal cholangiocarcinoma(d CCA),and(intrahepatic cholangiocarcinoma(i CCA).Among them,i CCA is a malignant tumor that occurs in secondary and above bile duct branch epithelial cells,accounting for about 20% of CCA.In recent years,the incidence of i CCA has been increasing year by year in China.Most patients are already in the advanced stage or terminal stage when they seek treatment,and the prognosis is extremely poor because of its relatively insidious onset and atypical early clinical symptoms.At present,the only possible way to cure i CCA is radical surgical resection.However,due to local invasion of advanced tumor,or presence of peritoneal,distant metastasis,or limited options for biliary duct reconstruction,only less than 1/3 of patients can undergo tumor surgery,the 5-year survival rate is only about30%,while postoperative recurrence rate is as high as 60% to 70%.Therefore,it is of great significance to explore the mechanism of the occurrence and development of i CCA and to find new early diagnostic markers and therapeutic targets for improving the prognosis of patients.Epithelial-mesenchymal transition(EMT)refers to the biological phenomenon in which epithelial cells lose their epithelial-cell characteristics and gain mesenchymal cell characteristics under certain physiological or pathological conditions.EMT process plays an important role in ontogeny,tissue regeneration,wound repair and organ fibrosis.In recent years,studies have found that EMT is involved in the malignant progression of various tumors.It is closely related to the occurrence,dryness and drug resistance of tumors,especially in the process of tumor invasion and metastasis.Therefore,reducing the occurrence of EMT in tumor cells is of great significance to reduce tumor metastasis and delay the malignant progression of tumor.According to the existing studies,the EMT process of cells is affected by many factors,among which deubiquitinating enzymes play a complex and important regulatory role.Ubiquitination refers to the process of ubiquitin modification of substrate protein residues under a series of catalytic enzyme joint action of ubiquitin-activating enzyme E1,ubiquitin-transferase enzyme E2,ubiquitin-ligase enzyme E3.Ubiquitination is one of the classic post-translational modification methods of proteins.The ubiquitination modification of proteins is a reversible process.Deubiquitinating enzyme(DUB)can specifically remove ubiquitin molecules from the substrate proteins,causing the substrate protein to deubiquitinate.The balance between ubiquitination and deubiquitination is an important foundation for maintaining stability and function of proteins in eukaryotes.Deubiquitinating enzyme is an important regulatory factor in cells,and its structural change,abnormal expression and uncontrolled activity are closely related to a variety of diseases including tumors.Studies have shown that a variety of deubiquitinases in the OTU-related proteases(OTUs)play an important role in the occurrence and development of malignant tumors such as lung cancer,gastric cancer,and colorectal cancer.In this study,we used clinical samples combined with database analysis to find that the expression level of deubiquitinating enzyme OTUB2 in i CCA tissues was significantly higher than that in paracancer tissues,and its expression level was correlated with the prognosis of patients with i CCA.A series of in vitro and in vivo experiments confirmed that OTUB2 can affect the proliferation,apoptosis,migration and invasion of i CCA cells.Further investigation revealed that deubiquitinating enzyme OTUB2 regulates the protein level of CTNNB1 in i CCA cells through coordination with E3 ubiquitination TRAF6,thus regulating the expression of EMT-driving factor ZEB1 in cells,and ultimately affecting the occurrence and development of i CCA.Objectives1.To investigate the expression level of deubiquitinating enzyme OTUB2 in i CCA.2.To analyze the relationship between the expression level of OTUB2 and the clinicopathologic features of patients with i CCA.3.To confirm the effect of OTUB2 expression level on i CCA cells.4.To elucidate the molecular mechanism of OTUB2 affecting the malignant progression of i CCA.Methods1.Using The Cancer Genome Atlas(TCGA)and Genotype-Tissue expression(GTEx)databases to analyze the difference of OTUB2 expression between normal and i CCA tissues.Immunohistochemical experiments and Western Blot experiments were carried out based on clinical surgical specimens and tissue chips,the results were further analyzed to verify the expression level of OTUB2 in i CCA and its relationship with the clinicopathologic features of patients.2.Lentiviral vector technology was used to establish human i CCA cell lines with OTUB2 knockdown.Western blot assay,CCK8 assay,single cell clonogenesis assay,scratch assay,Transwell assay and flow cytometry were used to verify the effect of proliferation and apoptosis,clonal formation,migration and invasion ability of i CCA cells induced by OTUB2 in vitro.3.The effect of OTUB2 on the occurrence and development of i CCA in experimental animals was investigated by constructing a mouse model of subcutaneous transplanted tumor and lung metastasis.4.Western Blot,Transwell and scratch tests were carried out using control and i CCA cell lines with OTUB2 knockdown,and database analysis was combined to explore the relationship between OTUB2 expression level and EMT-related proteins in cells.5.Chromatin immunoprecipitation,Western Blot,Transwell and scratch assay ware carried out using control and i CCA cell lines with OTUB2 knockdown,and the relationship between the expression level of OTUB2 and CTNNB1 protein was explored by database analysis.6.The specific molecular mechanism of OTUB2 promoting CTNNB1 protein expression in i CCA was elucidated by immunocoprecipitate,Western Blot and q PCR assay.Results1.The analysis of data from the Cancer Genome Atlas(TCGA)and Genotype-Tissue expression(GTEx)databases showed that OTUB2 expression was elevated in i CCA tissues and its expression level correlated with overall survival of patients.2.The expression level of OTUB2 in i CCA tissue samples obtained from clinical operations and i CCA tissue chips was found to be significantly higher than that in paracancer tissues,and its expression level was closely related to the pathological grade and clinical M stage of i CCA.3.In vitro,OTUB2 can enhance the proliferation and cloning of i CCA cell lines,reduce the apoptosis of i CCA cells,and promote the migration and invasion of i CCA cells.4.In vivo,OTUB2 can promote subcutaneous graft tumor growth of i CCA cells and lung metastasis.5.OTUB2 is positively correlated with the expression levels of EMT marker protein and EMT driving factor ZEB1 in i CCA cells,and can influence the EMT process of i CCA by regulating ZEB1 expression,thereby promoting the invasion and migration of i CCA cells.6.OTUB2 can increase the CTNNB1 protein level in i CCA cells,and affect the expression of ZEB1 through CTNNB1,thereby promoting the invasion and migration of i CCA cells.7.OTUB2 enhances CTNNB1 protein level by inhibiting CTNNB1 lysosomal autophagy.8.OTUB2 collaborates with E3 ubiquitination TRAF6 to regulate LC3 ubiquitination levels in i CCA cells,thereby regulating CTNNB1 autophagy.Conclusions1.The expression of deubiquitinating enzyme OTUB2 is elevated in i CCA and its expression level correlates with patient prognosis.2.OTUB2 can promote the growth,migration and invasion of i CCA cells in vitro and in vivo.3.OTUB2 can accelerate the EMT process by regulating the expression of EMT-driving factor ZEB1 in cells,thus promoting the migration and invasion of i CCA cells.4.OTUB2 can promote ZEB1 expression by regulating CTNNB1 protein level.5.OTUB2 and E3 ubiquitination TRAF6 collaboratively regulate the level of CTNNB1 protein in i CCA cells by regulating the level of LC3 ubiquitination.