Effects Of Gene-Environment Interaction And Tanscriptomic Changes During Precancerous Stages On Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is one of the most common gastrointestinal malignancies in China.The tumorigenesis of ESCC is a multistep process and the early precancerous stage has the ability to turn back into normal cells,suggesting the importance of early detection.Alcohol drinking and cigarette smoking are the known risk factors of ESCC.Besides the environmental factors,genetic factors and the gene-environment interactions also play important roles in the initiation and progression of ESCC.Previous genome-wide association studies(GWAS)have identified several single nucleotide polymorphism(SNP)sites affect the susceptibility of ESCC.Our ESCC GWAS also has identified risk SNPs located in alcohol dehydrogenase(ADH)gene family on 4q23.By performing stratified analysis,gene-environment interaction and eQTL analysis we identified the SNPs associated with ESCC susceptibility by interfering ADHs expression levels.To study the biological functions of target SNPs,dual-luciferase reporter assays and electrophoretic mobility shift assays(EMSA)were performed.In addition,ESCC mouse model was established using chemical carcinogens.Esophageal tissues of mice were collected in normal,inflammatory,mild dysplasia,moderate and severe dysplasia,carcinoma in situ and invasive cancer stage.We used 10x genomics to perform single-cell transcriptomic analysis of mouse esophageal tissues in different pathological stages to describe the dynamic molecular changes in the tumorigenesis of ESCC.We identified 8 eQTLs of ADH family that significantly associated with ESCC susceptibility(P<0.05).Dual-luciferase reporter assay indicates rs1154402-C allele has a significant higher expression level of reporter gene in compare with rs1154402-G allele(P=0.02).Besides,rs1154402-C allele has a significantly higher ability to promote the expression of ADH1A promoter,indicating a strong activity of enhancer.We performed interaction analysis to explore the effects of drinking status and genotypes of rs1154402 in ADH5 and rs11066015 in ALDH2 on ESCC susceptibility.We identified the odds ratio(OR)of drinkers with risk alleles of both SNPs are approximately 4-fold of nondrinkers.By performing pathway enrichment analysis,we found that immune activation related pathways are enriched in samples with high ADH1A expression level.By perforing single-cell RNA sequencing(scRNA-seq),we captured a total of 29,975 immune cells and 36,114 non-immune cell.Further analysis of 1,756 epithelial cells identified six sub-clusters and pathway analysis indicates different biological functions.Pseudotime analysis showed that epithelial cells showed two different evolutionary trajectories,one ended with terminal differentiation and the other ended with progression into malignant cells.Drinking is a vital risk factors of ESCC and ADH is an indispensable enzyme in the process of alcohol metabolism.As an extend study of previous ESCC GWAS,this study clarified that rs1154402,located in the intron of ADH5,is an enhancer which could regulate the expression level of ADH1A in a long-range mechanismand then finally affect the susceptibility ESCC.We also described the dynamic molecular changes during tumorigenesis of ESCC by scRNA-seq.We revealed six sub-clusters of epithelial cells and the composition of epithelial cells was different in each pathologic stages.We also systematically depicted the process of cell state transition in the ESCC tumorigenesis and identified epithelial cells showed two different evolutionary trajectories under the stimulation of persistent carcinogens.