The Basic Research And Clinical Significance Of The Expression Of USP22 And Ku80 In Esophageal Squamous Cell Carcinoma

Objective:Esophageal Carcinoma is one of the six most common malignant tumors in the world.Northeast Asia,Central Asia,South Africa and East Africa have high incidence of esophageal carcinoma.China has a high incidence of esophageal carcinoma,and squamous cell carcinoma accounted higher proportion compared with the esophageal adenocarcinoma in the foreign countries.With the extensive development and application of multidisciplinary treatment of esophageal carcinoma,such as immunotherapy of PD-L1,which was based on randomized,parallel,multicenter phase 3 clinical study,the diagnosis and treatment of esophageal carcinoma has gradually increased.However,due to high recurrence and metastasis characteristics of esophageal carcinoma,overall prognosis of patients with ESCC is still poor and the overall 5 year survival rate remained at 20%-30%.The prognostic factors of esophageal cancer,such as lymph node status,tumor differentiation and pathological staging,could not totally predict the recurrence and metastasis of esophageal carcinoma.Therefore,based on the genetic heterogeneity and heterogeneity of the tumors,it is urgent to seek the new markers which can predict the prognosis of ESCC and determine the high recurrence rate and metastasis of ESCC in different subgroups,and then determine individual and optimize therapy strategies.Ubiquitin specific protease 22(USP22)is a deubiquitinating enzyme which highly expressed in many cancers correlating with tumor recurrence and metastasis.Ku80 protein,a DNA binding protein,can bind DNA terminal and DNA double strand breaks(DSBs)which is induced by DNA damage.Recently some studies found that Ku80 plays an important role in the repair pathway on homologous binding end DSBs(NHEJ).In addition,Ku80 can also maintain telomere stability and regulate cell proliferation,apoptosis and signal transduction.Therefore,the purpose of this study was to explore the expression of USP22 and Ku80 in different pathological stages of ESCC and their relationships with the prognosis of ESCC patients by investigating the activation and interaction of USP22 and Ku80 in ESCC and evaluating the simultaneous model of USP22-Ku80 expression in esophageal squamous cell carcinoma.Methods:1.The expression of USP22 and Ku80 in esophageal cancer cellsThe subcellular distribution of USP22,Ku80 and ECA109 Ku80 were detected by immunofluorescence.Western blotting tested the different expression of USP22 and Ku80 in control ECA109 cells and overexpressed ECA109 cells.2.The expression of USP22 and Ku80 in ESCC tissuesA retrospective study collected 80 ESCC patients who had underwent esophageal resection and had complete follow-up data cases in the thoracic surgery of provincial hospital affiliated to Shandong University from January 2010 to December.Immunohistochemical method(IHC)was used to evaluate the expression of USP22 and Ku80 protein levels in ESCC tissues.2.Statistical analysis of the clinical pathological characteristics and prognosis in patientsThe clinical pathological characteristics and prognosis of patients were analyzed.In the study,we used t test to compare the relationship between proteins expression and pathological factors.Kaplan-Meier method and Log-rank test were used to analyze the postoperative survival.The analysis of continuous variables taken the mean standard and deviation of two variables was described by classification rate and percentage.To analyze whether there is a significant difference between the groups,single factor and multi factor variables were analyzed by COX regression analysis.Results:1.Higher expression of USP22 and Ku80 in esophageal cancer cellsThe subcellular distribution of USP22,Ku80 and ECA109 Ku80 were detected by immunofluorescence.The positive response showed red fluorescence which mainly distributed in the nucleus.The overlap method showed higher expression of USP22 and Ku80 in overexpressed ECA109 cells than these two proteins in the ECA109.Western blotting tested the different expression of USP22 and Ku80 in control ECA109 cells and overexpressed ECA109 cells.We also found that higher levels of USP22 and Ku80 in overexpressed ECA109 cells compared with the control ECA109 cells.2.USP22 and Ku80 expression were higher in tissues of ESCC patientsImmunohistochemistry(IHC)results showed that the positive expression of USP22 protein was mainly located in the nucleus of tumor cells,which showed yellow or brownish yellow staining,while there is no or light staining in the nucleus with negative expressed tissue.Ku80 showed the same pattern of USP22 in the immunohistochemistry.In addition,according to the results of immunohistochemistry above,all patients were divided into positive group and negative group,USP22 positive group(51 cases)and negative group(29 cases),Ku80 positive group(55 cases)and negative group(25 cases).At the same time,we analyzed the clinicopathological characteristics and prognosis of ESCC patients.The statistical results show that the expression level of USP22 was significantly correlated with the differentiation of tumor(P=0.008),T stage(P = 0.005).Moreover,the expression of Ku80 was also significantly correlated with tumor differentiation level(P=0.010),T stage(P=0.008).USP22 was significantly correlated with Ku80(P<0.001).What’s more,univariate statistical analysis of prognostic factors showed that the Overall Survival was significantly correlated with differentiation(P=0.001),T stage(P=0.023),N stage(P=0.005),NCCN clinical stage(P=0.009),the positive expression of USP22(P<0.001)and the positive expression of Ku80(P<0.001),Multivariate COX proportional hazards model analysis showed that the differentiation(P=0.029),USP22 positive expression(P=0.010)and Ku80 positive expression(P=0.002)were the independent prognostic indicators of overall survival(OS).The positive correlation coefficient between USP22 and Ku80 was 0.501,and the difference was statistically significant(P<0.001).The 1,3 and 5 year survival rates was 88.75%,45.00%,20.65%respectively in the total patients.The patients with USP22 positive was 82.35%,21.57%,and 9.80%,and the patients with USP22 negative was100%,82.76%,and 39.66%,respectively.Ku80 positive patients were 83.64%,27.27%,and 7.27%,while Ku80 negative patients was 100%,82.76%,39.66%respectively.Conclusions:1.The different expression of USP22 and Ku80 were found in human ESCC,and their expression levels were correlated with tumor differentiation and T stage.2.The expression of USP22 and Ku80 were closely related.3.The degree of differentiation,the expression of USP22 and Ku80 were the independent prognostic indicator of overall survival in ESCC patients.Innovations and significances:Therefore,as for ESCC,the expression model of USP22-Ku80 is expected to be a molecular marker for the diagnosis,prognosis and treatment of ESCC.