The Potential Interaction Of APE1 With ZEB1 And PD-L1 And Its Clinical Significancein Cervical Cancer

BackgroundCervical cancer is one of malignant tumor which threatensthe femalehealth in China.As far as we known,Relapse and metastasisof cervical cancerlead to the failure of threapy.EMT(epithelial to mesenchymal transition)plays an important role in the occurrence and development of cervical cancer.The important characteristics of EMT are loss of epithelial cell polarity and obtain the characteristic of mesenchymal cells.It is the main way to obtain migration ability.APE1 can not only repair DNA damage but alsoactivate many transcription factors viaits redox function..Furthermore,lymph node metastasis in patients haveincreased expression of APE1.This suggests APE1 is involved in the progress of cervical cancer.EMT is one of the important mechanisms in cancer metastasis,studies confirm APE1 participated in the regulation of EMT in non-small-cell lung cancer(NSCLC).However,whether APE1 regulatesEMT in cervical cancer and the regulation of the specific molecular mechanism is not fully clear.This study intends to use specific inhibitors and knockout cell lines,protein function locus mutation,CO-IP,migration of gel electrophoresis(EMSA)and immunofluorescence and nude mice experiment,aims to study theinvolvement of APE1 in EMT in cervicalcancer,eventuallyclarifiesthe mechanism of invasion and metastasis of cervical cancer and seeks the potential targets for interventionImmunotherapy shows a bright prospect in cervical cancer.Programmed death ligand 1(PD-L1)as a member of B7 family,is expressed by antigen-presenting cells,with functions of inhibiting activation of T lymphocytes.PD-L1,combined with its receptor induce T cell reactivity and/or apoptosis.Previous research found that APE1 and PD-L1 correlation in the expression,and respectively associated with tumor metastasis.However,there is not yet the literature system detailed reports of the relationship between the expression of APE1 and PD-L1 and the age,lymph node metastasis and prognosis in early cervical cancer.This study examined the PD-L1 and APE1 expression in early cervical squamous carcinoma tissues,analyzed the correlation andclinical significanceof PD-L1 and APE1,eventually providingclinical basis for the potential regulatory mechanism of APE1 and PD-L1.Objective1.To clarify whether APE1 participates in the regulation of EMT in cervical cancer,to explore specific molecular mechanisms of APE1 in EMT.2.To explore the APE1 and PD-L1 expression in early cervical squamous cell carcinomas correlation and the correlation between their level andclinical pathological factors.Methods1.The influence of APE1 and its redox function in EMT: Applying the methods of western blot,immunofluorescence,transwell,Scratch test and nude mice experiment to test APE1 knockdown and its redox deficient cell lines,aiming to investigate the regulatory effect of APE1 and its redox function on EMT.2.The mechanism of APE1 interact with ZEB1: Using theHA label of APE1 eukaryotic plasmid and FLAG label ZEB1 eukaryotic plasmid,APE1 Si RNA and APE1 REDOX function inhibitors-E3330,and applying the methods ofwestern blot,co-immunoprecipitationto unveil the interacting between APE1 and ZEB1.Using electrophoretic migration experiment(EMSA)to explore APE1 and it redox function can affect ZEB1 combining ability,influence of E-cadherin gene expression.3.Expression and clinical significance of APE1 and PD-L1 in the early stage cervical squamous carcinoma:Immunohistochemical staining was applied for the detection of APE1 and PD-L1 in 64 samples of early stage cervical squamous cell carcinoma.The correlation between the expression of APE1 and PD-L1 and clinicopathological features of patients with cervical cancer was analyzed.Spearman rank correlation was used to detect the correlation between APE1 expression and PD-L1 expression,Kaplan-Meier survival analysis was used to evaluate the impact of APE1 and PD-L1 expression on postoperative survival of the patients with early stage cervical squamous cell carcinoma.Cox univariate and multivariate prognostic analysis was utilized to explore the prognostic factors of the patients with early stage cervical squamous cell carcinoma.Results1.APE1 and its redoxfunctions can promote the EMT,and enhance the capacity of the metastasis of cervical cancer cells.APE1 can form protein complex with ZEB1,its redox functions does not regulate the expression of ZEB1,but reduce ZEB1 transcription activity and thus promote the expression of E-cadherin.2.The APE1 expression and PD-L1 expression were positive in 70.31% and 60.18% respectively.PD-L1 expression was closely correlated with FIGO stage and lymphatic metastasis(P < 0.05),and APE1 expression was closely correlated with lymphatic metastasis(P=0.038).Spearman rank correlation analysis indicated that APE1 and PD-L1 co-expression were positively correlated with the prognosis of early stage cervical squamous cell carcinoma(r2=0.347,P=0.005),and co-expression was closely correlated with lymphatic metastasis(P<0.05),on the other hand,the survival rates in both APE1 and PD-L1 positive expression group lower than those in either positive expression group(P <0.05).Multivariate Cox analysis revealed that the expression of APE1 and lymphatic metastasis played significant roles in the prediction of patient`s prognosis(P<0.05),Hazard ratio is 16.187(P=0.01)and 27.340(P<0.001)respectively.Conclusions1.In cervical cancer,APE1 combined with ZEB1 upregulate the transcription activity of ZEB1,reduce E-cadherin protein expression,promote EMT in cervical cancer.the process control dependson the APE1 REDOX function.2.APE1 expression associated with PD-L1 expression in early cervical squamous carcinoma,the higherAPE1 and PD-L1co-expression in early cervical squamous carcinoma patients have the poorer prognosis.These results illuminate that combinatorial measure of the expression of APE1 and PD-L1 has potentialin the diagnoses and prognosis of early stage cervical squamous cell carcinoma.