Explore The Potential Molecμlar Mechanism Underlying The Transformation Of Esophageal Epithelial Cells Into Esophageal Squamous Cell Carcinoma Caused By Nitrosamine And Gutter Oil Stress

The esophagus is composed of epithelial cells,epithelial stem cells and mesenchymal cells,in which epithelial cells are the most likely to contact with external stresses.When the layer of epithelial cells is destroyed,these external stresses will invade into the layer of stem cells,thereby destroying the microenvironment of epithelial stem cells and form esophageal cancer.Esophageal cancer is the fourth leading causes of cancer death in China.According to histological classification,esophageal cancer is divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma,esophageal squamous cell cancer(ESCC)is the major subtype of esophageal cancer accounting for more than 90%of esophageal cancer patients in china.ESCC is closely associated with mμltiple factors including the socio-economic status,lifestyle,habit factors such as drinking and smoking,and chemical,biological and genetic factors,which are the potential causes of esophageal cancer.Diethylnitrosamine(DEN)is a carcinogen which can be used as driver of esophageal cancer.Gutter oil is a complex which contains toxic substances such as polycyclic aromatic hydrocarbons(PAHs),which has potential carcinogenicity.In this study,these two chemical agents were used to treat esophageal epithelial cells and esophageal epithelial stem cells.Different levels of external environmental stress were applied to treat esophageal epithelial cells and esophageal stem cells to simμlate the stress response of normal esophagus to these stresses.Then the changes of gene expression patterns and epigenetic regμlation patterns caused by these stresses were comprehensively analyzed.Comprehensive analysis was performed based on the resμlt of methylation detection and RNA sequencing,23 genes with high methylation and low methylation of interest in were found,such as GDA,ONECUT2,RASAL1 and so on.Meanwhile,we used real-time quantitative PCR to detect the expression patterns of 23 genes of interest in two esophageal epithelial cell lines(HEEC and HET1A)and several esophageal squamous cell carcinoma cell lines(KYSE30,KYSE150,KYSE450 and TE-15).In addition,the stressed esophageal epithelial cells possessing stemness were cμltured in 3D cells,and the malignant status of these epithelial cells was evaluated.Finally,ONECUT2 was determined for further study after bioinformatics analysis.Tissue microarray was used for detecting the expression pattern of ONECUT2 in clinical esophageal squamous cell carcinoma cohorts,and the resμlt showed that the protein level of ONECUT2 is significantly higher in esophageal cancer tissues when comparing with their adjacent tissues,and the high level of ONECUT2 in esophageal cancer tissues is closely related to the occurrence of esophageal cancer.To analyze the function of ONECUT2 in ESCC,KYSE450 cell line with ONECUT2 knock out was established for subsequent function assay.In this study,the relationships between gutter oil and esophageal squamous cell carcinoma,and the transformation of epithelial stem cells into esophageal squamous cell carcinoma caused by nitrosamine and gutter oil were preliminarily determined,and the carcinogenic evolution of esophageal epithelial stem cells was also studied...