Comparative Study On The Molecular Characteristics Of Human Esophageal Squamous Cells Carcinogenesis

Chapter 1:Comparative genetic analysis of oesophageal dysplasia and squamous cell carcinoma reveals the evolutionary landscape of ESCCBackground:Esophageal squamous cell carcinoma(ESCC)is a malignant tumor of the digestive tract with a high mortality rate.With the development of high-throughput sequencing technology,various genetic changes leading to ESCC have been widely revealed,but to apply the current research results to guide the early diagnosis and treatment of ESCC,it still requires further clarifying the selection and sequence of various genetic changes during multiple stages of the carcinogenesis process,as well as the evolution of the entire process of esophageal carcinogenesis.Severe dysplasia is a recognized precancerous lesion of ESCC.A comparative study of corresponding genetic changes of severe dysplasia and ESCC can help to further refine distribution of various genetic variants over the entire time axis of esophageal carcinogenesis.Objective:This project aims to explore the selection of various types of genetic changes in multiple stages of esophageal carcinogenesis and the sequence of their occurrence by comparing and studying the differences of various types of genetic changes between precancerous lesions and ESCC tissue,and to summarize the evolutionary law of the occurrence and development of the process,and try to find the events at several nodes such as the beginning of the esophagus carcinogenesis and the transformation from precancerous lesion to cancer.Materials and methods:In this study,a total of two types of esophageal tissue cases,temporal longitude and spatial latitude,were selected for whole exon sequencing,and various genetic changes such as SNV,LOH,CNV,and WGD were analyzed and compared Temporal longitude cases are different lesions sampling at similar anatomical locations at different time nodes of the same patient,including 4 patients with 4 adjacent normal epithelial tissues,6 dysplasia epithelia tissues,and 4 squamous cell epithelial tissue samples in total.Spatial latitude cases included severe dysplasia samples adjacent to cancers and ESCC tissues taken at the time point diagnosed as ESCC.A total of 23 patients with 23 adjacent normal epithelial tissue,23 adjacent severe dysplasia epithelial tissue,and 23 ESCC tissue samples.Results:We used temporal longitude cases as typical cases,and spatial latitude cases as an expanded case group to compare the genetic changes of precancerous lesion and ESCC genome.It was found that there was a significant difference in genetic changes between precancerous lesions and ESCC.There was still some consistency in genetic changes.On LOH,it occurs relatively concentrated on specific chromosomes in both precancerous lesion and ESCC samples.In paired precancerous lesion and ESCC samples,the degree and location of LOH have a high consistency.On SNV,there is a significant difference in the number of mutations between precancerous lesions and cancer,but most of the precancerous and cancer pairs have comparable shared mutations.The mutations of tumor suppressor genes such as TP53 and CDKN2A generally occur early with the highest consistency between precancerous and ESCC genome.On CNV,precancerous lesions and ESCC share 3q,8q22.4,11q13.3 and other high-frequency CNV regions containing well-known oncogenes,but there are rare other shared CNV between precancerous lesions and cancers.And the number of CNV surged in ESCC compared to severve dysplaisa.WGD rates are very different in precancerous and ESCC tissues in longitudinal samples,but they have a very similar incidencein two types of samples of spatial latitude cases.Based on the similarities and differences of various genetic variants in ESCCand precancerous lesion,we inferred the evolutionary relationship between the paired precancerous lesion and cancer samples.We found that there are 3 different types of precancerous lesion to ESCC evolution traits in both the longitudinal and spatial cases.The main evolutionary trait of ESCC are the closest one among the three.Conclusion:The similarities and differences of different types of genetic variation in precancerous lesion and ESCC indicate that on the time axis of esophagus carcinogenesis,different types of genetic changes occur at a selective stage,LOHs concentratedly occur early during the carcinogenesis process;SNVs are accumulated uniformly throughout the entire process of carcinogenesis.The mutations obtained at different stages are related to the biological behavior of the lesion at relative stage.The mutation of TP53 and other tumor suppressor genes are important events for the onset of ESCC;CNV mainly affects malignant development of carcinogenesis process,but the amplification of a small number of oncogenes are an early event of esophageal carcinogenesis;WGD is an important genetic change at the node of cancer transformation from a precancerous lesion.Three evolutionary distance between the precancerous lesions and cancer were observed in esophageal epithelium.ESCC with both TP53 mutation and WGD are more likely to have a closer evolutionary relationship with their precancerous lesions.Chapter 2:Comparative study of precancerous lesions and squamous cell carcinoma transcriptome and the study of the abnormal expressed LINC01605 during the esophageal carcinogenesis.Background:Severe dysplasia of the esophagus is an important precancerous lesion of ESCC.Single-molecule comparative studies and a small number of comparative exon sequencing have revealed that the two pathological states of esophageal epithelium have many similarities at the molecular level.However,researchers have paid relatively little attention to the molecular difference of precancerous lesions and ESCC.At the same time,at the transcription level,there is still a lack of comprehensive studies to reveal the differences betweenprecancerous lesions and ESCC expression profiles from the perspective of omics.Fully revealing the similarities and differences between esophageal precancerous lesions and ESCC at the transcriptome level,combined with the similarities and differences at the genomic level,can better understand the carcinogenesis process of ESCC.Objective:This part of the study aims to explain the transcriptome changes at different stages of esophageal carcinogenesis by comparing the differences in the expression profiles of esophageal precancerous lesions and ESCC,and explore the important molecular changes that promote the progression of precancerous lesion transformation to ESCC.Materials and methods:First of all,the expression profile data of 12 normal tissues,5 severe dysplasia,5 severe dysplasia marginal normal tissues,and 7 ESCC tissues from 5 severe dysplasia patients and 7 ESCC patients were completed in the previous laboratory work.Expression level of genes were compared among groups to find genes that were differentially expressed between groups and analyze related functions.Then focus on the genes that only change between precancerous lesion and ESCC,look for genes that play an important role in precancerous lesion transformation to ESCC,and use a variety of molecular biology methods to explore the influence of candidate genes in vitro on phenotype of ESCC and related mechanism.Results:comparisons of normal,severe dysplasia and ESCC identified a total of 2345 differentially expressed genes.At the level of transcription,severe dysplasia that has not progressed to ESCC is already very similar to ESCC on the behaviors of unlimited replication capacity,self-obtained growth signals,consistent anti-growth signals,and genomic instability and mutations.In terms of energy metabolism remodeling,apoptosis escape,and continuous angiogenesis,severe dysplasia samples have obvious differences in cancer,even less than normal samples.The aspects of tumor invasion and metastasis,tumor promotion of inflammatory response,and tumor immune escape have been slightly reflected in severe dysplasia,but they are still significantly different from states in ESCC.Through co-expression analysis,we identified a co-expression module containing 49 genes in ESCC that are only different expressed between severe dysplasia and ESCC.The expression of this module genes has an influence on patient prognosis.Through CCK-8 and Transwell experiments,it was proved that the expression of LINC01605 gene,a lncRNA in this module can inhibit the growth and migration of esophageal immortalized epithelial cell line and ESCC cell lines;RNA pull down experiment proved an interaction of this lncRNA and AURKB protein.Conclusion:There are significant differences in the transcription level of normal esophageal tissue,severe dysplasia,and ESCC.Severe dysplasia has already possessed part of the tumor hallmarks,but a considerable part of the tumor hallmarks were acquired during the process of precancerous lesion progressing to ESCC.Differently expressed genes of different dynamic modes play different functions in the process of carcinogenesis,and abnormal expression of genes with related function such as angiogenesis,cell adhesion and metabolic are concentratedly dysregulated during the progression from precancerous lesion to ESCC which plays an important role in the later stage of ESCC progression.During the progression from precancerous lesion to ESCC,a 49 co-expressed gene module can indicate tumor prognosis.The gene LINC01605,which is low expressed in ESCC in this module,can inhibit the growth and migration of tumor cells and may affect cell growth through the interaction between LINC01605 and AURKB protein.