A Preliminary Study On The Possible Association Between Helicobacter Pylori And Esophageal Carcinogenesis

Background and objective Esophageal cancer(EC)is one of the most common malignant tumors of digestive tract in China.The incidence rate is about 7.1% of all new tumors in China,and the mortality rate is about 10%of all tumors in China.Histologically,it can be divided into Esophageal adenocarcinoma(EAC)and Esophageal squamous cell carcinoma(ESCC).Initial EC has no obvious clinical symptoms.When there are obvious progressive dysphagia symptoms,it usually has entered the middle or late stage.So the patient’s quality of life is greatly reduced.Therefore,to find the risk factors related to esophageal cancer is of great significance for the prevention and treatment of EC.Helicobacter pylori(H.pylori)is a gram-negative pathogenic bacteria that can be colonized in the stomach and cause a variety of diseases,such as peptic ulcer,chronic gastritis,stomach cancer and stomach lymphoma.However,the relationship is not obvious between the H.pylori infection and the esophagus disease.A negative association between H.pylori infection and EAC was reported to be a protective factor for EAC.However,studies in high risk areas of EC in China suggest that H.pylori infection is a high risk factor for ESCC.Therefore,the purpose of this study is to explore the effect of H.pylori infection on the occurrence and development of ESCC through in vivo and in vitro experiments,so as to provide a new idea for the prevention and treatment of ESCC.Materials and MethodsMaterials1.Tissue samples: postoperative cancer and cancer tissue samples from 50 ESCC patients without radiotherapy and chemotherapy were collected from the Department of Pathology,Cancer Hospital of Shantou University Medical College.According to historical classification,the specimens were divided into normal epithelium(n=50),intraepithelial neoplasia(n=38)and ESCC(n=50).At the same time,DNA was extracted from 15 cases of normal epithelium and 13 cases of cancer tissue..2.Cell line and Bacterial strain: NE6 esophageal immortalized cell line was presented by Professor Cao Shihua of the University of Hong Kong.H.pylori strain was isolated and cultured by our team.Methods1.Detection of H.pylori infection in patients with esophageal cancer: 28 cases of esophageal tissue samples(15 cases of distal margin mucosa and 13 cases of cancer tissue)were amplified by polymerase chain reaction(PCR),and the PCR results were verified in paraffin specimens by fluorescence in situ hybridization(H.pylori probe).2.Effects of H.pylori strain on NE6 cells: The strains of H.pylori were co-cultured with NE6 cells in vitro,and the effects of H.pylori on NE6 cell morphology were observed by special staining and microscopy.The effects of H.pylori on NE6 cell proliferation were detected by CCK-8,and the expressions of 8-OHdG and 53BP1 in NE6 cells were detected by immunofluorescence.The effect of H.pylori on the m RNA transcription level of NE6 cells was detected by transcriptome sequencing and the differentially expressed genes were screened.3.Detection of the expression of differentially expressed genes in the process of esophageal carcinogenesis: the expression of differentially expressed gene,apoptosis inhibitory protein 3(BIRC3),in different stages of esophageal carcinogenesis was detected by IHC.Results1.There are 6 of the 28 ESCC samples that were positive for the CagA gene,and fluorescence signal of H.Pylori was detected in four patients by FISH.2.After co-culture,H.pylori can be seen around and in the spaces between the cells.Compared with the non-co-cultured group 8-OHdG,the scattered expression in the 53BP1 nucleus became focal aggregation.In addition,different strains can promote cell proliferation at different degrees.3.After co-culture,the transcription level of NE6 cells also changed,a total of 788 shared DEGs were identified,including 508 up-regulated and 284 down-regulated(| log2(Fold Change)| > 2,padj< 0.001).By enriching the functions and pathways of the DEGs,the up-regulated DEGs were mainly enriched in response to decreased oxygen levels,apoptosis,leukocyte activation,mainly enriched in tumor and inflammation pathways;down-regulated DEGs were mainly enriched in keratinization,type I interferon signal pathway,negative regulation of JUN kinase,mainly enriched in a variety of amino acid metabolic pathways.For Disease Ontology(DO),the DEGs were mainly enriched to tumors and inflammatory diseases,and could be enriched in esophageal carcinoma.4.The expression of BIRC3 was increased both in esophageal intraepithelial neoplasia mucosa and ESCC tissues compared to the para-cancerous normal mucosa.ConclusionsH.pylori infection can be present in esophageal tissue.H.pylori can adhere to the cell surface when infected NE6 cells,cause cell morphological changes,promote cell proliferation,cause DNA damage,and extensively change the gene transcription level.These differential genes are mainly involved in the response to oxygen level decline,apoptosis,intercellular adhesion,white blood cell activation,angiogenesis and other functions.