Esophageal Squamous Cell Carcinoma Of The Differentially Methylated Fragment Screening And Identification Of Genes

Esophageal carcinoma is one of the 10 most common malignant tumors in human, which accounts for 1.2 million new cases worldwide annually and most of them occurred in developmenting countries, meanwhile it claims about 330,000 lives worldwide each year. In China, esophageal cancer is the sixth most common cancer; meanwhile, it ranks as the fourth leading cause of cancer-related death, accounting for 150,000 deaths per year. The difficulty of early diagnosis contribute largely to the high mortality of esophageal cancer, for example, patients with early-stage esophageal cancer always have a good clinical outcome, whereas the overall 5-year survival is only about 10% in the late or advanced stage of ESCC. Therefore, further researches of screening new marker for early detection and developing new cancer treatment remain the best hope for a cure. Many factors were involved in the development of esophageal carcinoma, among them environment stimulus has been proved to be a high risk factor. During recent years, there has been a growing interest in the role of epigenetic changes in carcinogenesis, especially DNA cytosine methylation in gene promoter regions, which was the relatively early and stable reaction to long-term environment stimulus, therefore DNA methylation may be of great value in tumor early detection. Except that, as the methylated genes always play important factors in cell development and regulation and the epigenetic silencing can more or less be reversed by drug treatment, study of epigenetic modification shed light on new field in tumor therapy. Compared with numerous studies in other tumor types, it is being in the primary stage for ESCC epigenetic modification. Therefore, screening ESCC-related hypermethylated fragments and exploring their functions in tumorigenesis will significantly contribute to the further studies of tumor molecular mechanism, early detection measures and novel effective therapies.Objects: To screen novel tumor-related genes being aberrantly hypermethylated and study functions of those genes in the progression of ESCC, then go further to provide basement in the further research of early detection and effective therapeutic strategies in ESCC.Methods: Firstly, differentially methylated CpG islands were identified between several paired ESCC tumors and normal tissues by methylation-sensitive AP-PCR (MS AP-PCR) method, which were visualized as bands in the electrophoresis gels after silver staining, and then those fragments screened were isolated and subjected to bioinformatic analysis. Subsequently, the functions of those meaningful fragments were, in ESCC tumor tissues and cell lines, investigated with several methods including bisulfite-sequencing PCR(BSP), RT-PCR and Quantitative real time PCR (QRT-PCR), to explore their potential roles in the Carcinogenesis and Progression of ESCC.Result:(1) Identification of differentially methylated DNA fragments in ESCCDifferently methylated maps of ESCC were obtained by screening paired tissues with genome-wide methylation analysis method. Twelve hypermethylated DNA fragments (HMDF) were identified, and seven of them containing the GC-rich sequences conformed to the minimal criteria that define CpG islands. Within those seven fragments containing CpG islands, five of them were located in the 5' region of known genes; meanwhile, all of them contain several transcript factor binding sites.(2) Further studies of meaningful fragments identified in ESCC2.1. Aberrant methylation of several corresponding genes in ESCC2.1.1 Aberrant promoter methylation of the TPEF geneMethylation status of TPEF gene promoter and expressions of TPEF transcript were detected in 22 ESCC samples, in which 12 (54.5%) of 22 ESCC tumors exhibited TPEF promoter hypermethylation accompanied by reduced expressions when compared with matched adjacent normal tissues. Studies on ESCC cell lines showed decreased methylation status of TPEF gene promoter and restored expressions of TPEF mRNA after treatment with the demethylating agent.2.1.2 Aberrant promoter methylation of the EDNRB geneMethylation status of EDNRB gene promoter and expressions of EDNRB transcripts were detected in 21 ESCC samples, in which 5 (23.8%) of 21 ESCC tumors exhibited EDNRB promoter hypermethylation and reduced expression occurred compared with matched adjacent normal tissues. Studies on ESCC cell lines showed decreased methylation status of EDNRB gene promoter and increased expression levels of EDNRB mRNA after treatment with the demethylating agent.2.2 The preliminary study of a large region being methylated in ESCCAn interesting result was that two HMDFs (HMDF 12 and HMDF3) are both located at chromosome 13q22.3, the regions between them spanned about 1.5Mb and contained several important genes. Studies on ESCC cell lines showed that the mRNA levels of almost all genes located at the region were increased in different degrees after treatment with the demethylating agent.Conclusions:In our study, ESCC special genome-wide different DNA methylation profiles and meaningful hypermethyled fragments were obtained by MS AP-PCR, and it was the first report about aberrant hypermethylation of TPEF and EDNRB genes in ESCC. Meanwhile, we firstly explored a large hypermethylated region in ESCC, genes located in which were epigenetic silenced and mRNA expressions could be restored by treatment with the demethylating agent.