The Role Of DNA Methylation Of Candidate Gene SPATA32 And ZNF132 In The Diagnosis Of Esophageal Cancer And The Study Of The Function Of ZNF132

Esophageal cancer is one of the common digestive tract malignant tumor.In recent years,the incidence of esophageal cancer is on the rise,in our country,esophageal cancer has become the fourth big cancer deaths,which harms people’s health seriously.Esophageal cancer can be divided into Squamous Cell Carcinoma(ESCC)and Esophageal Adenocarcinoma(EAC),and China is ahigh incidence area of Esophageal cancer,mainly of Squamous Cell Carcinoma.The mortality of esophageal cancer is high,the treatment effect is not good,year survival rate is less than 10%,so the screening of biological indicators of early diagnosis and the research on the transcription factors that regulate the evolution of tumor and the methylation of its regulatory gene are closely watched by the researchers.This experimental study is carried out from the following two parts:Part one:we analysis illumina450K methylation of microarray data from TCGA website(https://cancergenome.nih.gov/)database,obtained 7 candidate hypermethylaton gene sites in esophageal cancer,then we tested these candidate genes in 94 cases of esophageal cancer tissue and paired the para-carcinoma tissue by bisulfite sequencing,and the results showed that out of 7 candidate genes the gene ZNF132、ZNF470、SPATA32 are in a hypermethylaton situation in the tested esophageal cancer tissue.We analyzed predictive ability of the above 7 candidate genes for early diagnosis of esophageal cancer and set up Multi-gene joint diagnosis model organized by gene ZNF132、ZNF470、SPATA32 aiming to get better forecast effect,we also test the predictive ability of the diagnosis model by statistical analysis approach as Logistic RegressionRandom Forest、SVM、Naive Bayes、Neural Network、Linear Discriminant Analysis、Mixture Discriminant Analysis、Flexible Discriminant Analysis and Gradient Boosting Machine.Among the 7 candidate gene,ZNF132 has best predictive effect with its AUC value is 0.83,but its specificity is 81%not as good as that of gene SPATA32,then we compare the predictive effect of Multi-gene joint diagnosis model with ZNF132,the results showed the AUC value of the diagnosis model is still 0.83 the same as ZNF132,the specificity increased,but the sensitivitydecreased which indicate that the prediction effect of the multi-gene joint diagnostic model was similar to that of single gene ZNF132,without significant improvement,and it also means the methylation situation of ZNF132 can be used as a biomarker for early diagnosis of esophageal cancer with potential clinical application value,so it has great importance to explore the molecular regulation mechanism of ZNF132 in the development of esophageal cancer.Part two:we carried out certain experiments to explore the regulation of ZNF132 methylation situation,the biological functions of ZNF132 gene and the regulation of ZNF132 gene expression in esophageal cancer.The results showed:1.In vitro,the control sequence of ZNF132 gene was treated with methylation and demethylation,showing that the gene expression was regulated by methylation modification.2.By observing the influence of ZNF132 on the biological behavior of esophageal cancer cell proliferation,apoptosis and migration ability,and its effect on the tumor capacity of the tumor mice,we identified ZNF132 plays a role in inhibiting tumor cell proliferation,migration and invasion,significantly promotes the apoptosis of tumor cells and inhibits the formation of subcutaneous tumors in mice as a tumor suppressor gene does.3.Using the dual-fluorescence reporting system,chip-PCR analysis and DNA Pulldown and other experiments to analyze the regulation mechanism of transcriptional activator Spl on ZNF132 expression,the results show that Spl can be combined with ZNF132 promoter sequence at the Sp1 binding site and the methylation of this binding site can affect the ability of Sp1 to bind ZNF 132 at this promoter region,resulting in ZNF 132 gene expression decreasing.In this study,the feasibility of the methylation of ZNF 132 gene as a biological marker was determined,which provide new ideas and methods for diagnosis of esophageal cancer.In addition,a further analysis of gene ZNF 132 function was established to lay a foundation for the molecular mechanism in the development of esophageal cancer.