Preliminary Study On The Expression Of IgA Subtypes And Basement Membrane Related Components During The Carcinogenesis Of Esophagus

Background and purposeBackground:Esophageal cancer is one of the most common malignancies in the world.According to the latest global epidemiological survey,there will be an estimated 604,000 new cases of esophageal cancer worldwide in 2020,ranking seventh in incidence and sixth in mortality,it is estimated that about 544,000 patients died of esophageal cancer.Compared with the statistics in 2018,the number of new cases and patients who died from esophageal cancer have increased.It is worth noting that Chinese people are still the majority of esophageal cancer patients.Until2015,the incidence of esophageal cancer ranked third and fifth among Chinese male and female cancer patients,respectively.Related mortality ranks fourth among all malignant tumors.Esophageal cancer is also highly prevalent in the Chaoshan area in eastern Guangdong Province,China,especially on Nan'ao Island.Therefore,studying the pathological changes of the esophagus at each stage of carcinogenesis will have important value for the diagnosis and treatment of esophageal cancer.Purpose:Firstly,immunohistochemistry(IHC)staining was used to observe the expression differences of IgA1,IgA2,basement membrane and hemidesmosome components(laminin-?2,integrin-?4)in different stages during esophageal carcinogenesis(normal,simple hyperplasia,dysplasia,invasive carcinoma).Secondly,the transcript sequencing data of the different stages of esophageal carcinogenesis of our group and the normal esophageal transcript sequencing data from the GTEx database were analyzed for immune cell subpopulations;meanwhile,the expression profiles of IgA,basement membrane components,MMP,and reactive oxygen species synthase-related genes were utilized for differential expression analysis.Finally,the mouse esophageal cancer model constructed by our groupmates was used to observe the ultrastructure changes of basement membrane,hemidesmosomes and the expression changes of laminin-?2 and integrin-?4 by transmission electron microscopy and IHC staining;at the same time,in immortalized normal esophageal epithelial cell line NE6,the effects of reactive oxygen species stimulation on cell proliferation,migration,and expression of laminin-?2 and integrin-?4were observed.Materials and methods Materials1.Sample collection:This study collected 189 specimens from 73 patients with esophageal squamous cell carcinoma(including the distal esophageal mucosa,adjacent esophageal mucosa and cancer tissues)and 26 normal esophageal tissue samples from autopsy;2.Transcript sequencing data includes data of 80 normal esophageal mucosal tissues from GTEx database,as well as 72 esophageal samples from different cancerous stages that have been sequenced by our group(including normal,hyperplasia,dysplasia and cancer tissues);3.C57 mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co.,Ltd.and NE6,a normal esophageal squamous epithelial immortalized cell line was gifted by Prof.George Tsao from the Department of Biomedicine of the University of Hong Kong.Methods1.In the current study,the paraffin sections of the esophageal tissue samples were used to observe the cell morphology,assess the type of lesion and the level of inflammation through HE staining;use immunohistochemical(IHC)staining to detect CD38,IgA,IgA1,IgA2,integrin-?4 and laminin-?2 protein expression in the esophageal tissue;2.Using the transcript sequencing data of esophageal tissue to analyze the composition of immune cells in the sample through CIBERSORT,and use Edge R to analyze the differential expression of genes between each group;3.Using the C57 mouse esophageal carcinogenesis model constructed by our group,HE and IHC staining were used to observe morphological changes during the process of esophageal carcinogenesis,as well as changes in the expression of IgA,laminin-?2 and integrin-?4;observing the ultrastructure of hemidesmosomes by transmission electron microscope;at the same time,by engaging H₂O₂ and 4NQO treatment to NE6 cell line,wound healing assay,CCK8,clone formation assay,immunofluorescence staining,western-blot were utilized to observe the cell proliferation and migration ability,and laminin-?2,integrin-?4 protein expression.Results1.The results of IHC staining and transcript sequencing data show that the expression of IgA1 in normal esophagus and different cancerous stages(normal,simple hyperplasia,dysplasia,invasive cancer)tissues are higher than IgA2,and the ratio of IgA1 to IgA2 is significantly increased in invasive cancer tissue;the expression of IgA,IgA1,IgA2,laminin-?2 and integrin-?4 increased with the progression of cancer and the severity of inflammation;2.In tissues at different stages of esophageal carncinogenesis,the damage of basement membrane is accompanied by the infiltration of IgA-positive inflammatory cells,neutrophils,and the expression of laminin-?2 and integrin-?4;at the same time,the transcripts of tissues at different stages of esophageal cancer are sequenced.Data analysis show that the transcription levels of IGHA1,IGHA2,LAMC2,and ITGB4 genes are positively correlated with the stage of esophageal canceration,plasma cell content,and neutrophil content;3.In invasive cancer tissues,IgA and IgA1 are positive for IHC staining of cancer cells at the edge of the cancer nest;laminin-?2 and integrin-?4 can be expressed in cancer cells at the edge of the cancer nest or all cancer cells;4.The hemidesmosomes in the dysplastic esophagus tissues of mice are malformed and unevenly distributed;the stimulation of reactive oxygen species promotes the proliferation of NE6 cells,up-regulates the protein expression of laminin-?2 and integrin-?4,and the localization of integrin-?4 from the cell membrane to the cell membrane.Changes in cytoplasm.Conclusions1.The expression of IgA1 and IgA2 in cancerous tissues of the esophagus is significantly higher than that in normal tissues,and IgA1 is the dominant subtype in esophageal tissues.Cancer cells at the edges of cancer nests are positive for IHC stainig against IgA and IgA1;2.The transcription levels of IGHA1,IGHA2,LAMC2 and ITGB4 genes are positively correlated with the composition of plasma cells and neutrophils in esophageal precancerous lesions;3.The destruction of the ultrastructure of basement membrane and hemidesmosomes is an early event of esophageal carcinogenesis.The chronic esophageal inflammation microenvironment may play a certain role in promoting the changes of hemidesmosome structure and related protein expression in esophageal precancerous lesions.