| Esophageal cancer (EC) is a malignant disease of esophageal mucosa and esophageal epithelial. There are two different types of histopathology:esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Tumor pathogenesis and pathological outcomes of two different types of histopathology are different. Wordwide, the incidence and mortality of the esophageal cancer are the ninth and the eighth in all the malignant tumors. The global statistical report of International Agency for Research on Cancer (IARC) shows that an estimated482,300new esophageal cancer cases and406,800deaths occurred in2008worldwide. The characteristical distribution of esophageal cancer is regional distribution. Stretching from northern Iran through the central Asian republics to North-Central China, often referred to as the "esophageal cancer belt,"90%of cases are squamous cell carcinomas.In the year2008, in China there are259,000new cases of esophageal cancer of the1.34billion Chinese mainland population. The incidence rate of esophageal cancer is16.7/104which ranks the fifth in all types of cancer.211,000deaths occur due to esophageal cancer and the mortality rate is13.4/104which ranks the fourth in all types of cancer. There are176,000new male esophageal cancer patients and144,000deaths, the incidence is22.9/104and the mortality rate is18.7/104of male, there are83,000new female esophageal cancer patients and67,000deaths, the incidence is10.5/104and the mortality rate is8.2/104of female. The incidence of esophageal cancer of male is the fourth in all the malignant tumors and the incidence of esophageal of cancer of female is the seventh in all the malignant tumors. Both the mortality rate of esophageal cancer of male and female are the fourth in all the malignant tumors.In China, the typical epidemiological characteristic of esophageal cancer is the difference between different areas. The incidence of esophageal cancer is sharply higher in the center area contrast to the surrounding areas. The incidence of esophageal cancer is higher in provinces nearby the Taihang Mountains and Linzhou country has the highest incidence of esophageal cancer in China. Therefor, in our contry esophageal cancer has become a public healthy problem which endanger people’s health and lives, particularly in the areas in which the incidence of esophageal cancer is higher.However, the etiology of esophageal squamous cell carcinoma is not yet fully understood. Epidemiological studies have found that unblanching diet, nutritional deficiencies, drinking beverages at high temperatures, eating sauerkraut and nitrite contamination food, mycotoxins, smoking, drinking play important roles in the pathogenesis of esophageal squamous cell carcinoma. Nevertheless, only a few part of individuals who esposed to the same risk factors become esophageal squamous cell carcinoma patients. In addition, in the areas of high indigence of esophageal squamous cell carcinoma, the incidence of esophageal squamous cell carcinoma exhibits familial aggregation. These phenomena argue that there is an interaction of environmental factors and genetic factors and both of them play important roles in the pathogenesis of esophageal squamous cell carcinoma. Therefor, it is imperative to further understand the underlying molecular mechanism of esophageal squamous cell carcinoma. In addition to genetic factors, epigenetics (especially DNA methylation) also paly an important role in the pathogenesis of esophageal squamous cell carcinoma. Importantly, epigenetic changes, including alterations in DNA methylation, are reversible and can thus be targets for cancer therapy or chemoprevention. Therefore, a better understanding of the DNA methylation in esophageal squamous cell carcinoma is important for optimizing cancer therapy and chemoprevention.Surgery is one of the main effective means of treatment for esophageal squamous cell carcinoma. Despite significant advances in diagnosis and treatment of esophageal squamous cell carcinoma, the five year survival rate is still about10%because of its asymptomatic progression in the early stage. The majority of esophageal squamous cell carcinoma patients has advanced metastatic disease at initial diagnosis and is inappropriate for curative resection. In sharp contrast, the five-year survival rate for ESCC patients with endoscopic mucosectomy at early stages, i.e. carcinoma in situ and intramucosal carcinoma, was100%after endoscopic mucosectomy, and surgery gives an overall five-year survival rate of90%. Early diagnosis is an important factor in determining5-year survival rate of esophageal squamous cell carcinoma. The research reports that in the eraly stage of the cancer there is alteration of DNA methylation and these abnormal DNA methylations can be detected in body fluids and early lesion biopsies. What’s more, DNA methylation can be restored by demethylating agent, suggesting that abnormal DNA methylation may be as biomarkers for early diagnosis and targets of biological target therapy.In the current study, we analyzed global methylation profiling of esophageal squamous cell carcinoma and normal adjacent tissue in Chinese cancer patients and esophageal mucosa from Chinese healthy individuals, using Infinium Methylation450K array. After analysis of the methylation differences and then in combination with independent gene expression data using BRB (Biometrics Research Branch)-Array Tools of the National Cancer Institute, a set of genes that are deregulated by aberrant DNA methylation in esophageal squamous cell carcinoma was identified. We then focused on2aberrant DNA methylation genes—EPB41L3and GPX3, with validation analysis using additional esophageal squamous cell carcinoma tissues and adjacent normal tissues. In order to evaluate whether the methylation status of the2candidate genes is useful for diagnosing ESCC, we also tested the methylation of the circulating cell-free DNA in patients’ plasma and controls.Our research including three parts:The first part:Objective:To analyze the whole DNA methylome of esophageal squamous cell carcinoma in Chinese patients. Methods:We analyzed global methylation profiling of esophageal squamous cell carcinoma tissue and normal adjacent tissue in Chinese cancer patients and esophageal mucosa from Chinese healthy volunteers, using Infinium Methylation450K array. We imported the chip raw data into BRB-array Tool (4.3.2version) for data analysis. Results:Methylation chip raw data clustering analysis and multidimensional scaling analysis showed that the2main clusters were each composed by a phenotype:tumor and normal. Difference analysis of gene sets found that multiple signaling pathways are significantly difference between esophageal squamous cell carcinoma and control normal esophageal mucosa. Conclusion:Esophageal squamous cell carcinoma and normal esophageal mucosa have a different DNA methylation pattern. DNA methylation abnormalities may play an important role in the tumorigenesis of esophageal squamous cell carcinoma.The second part:Objective:To screen the potential functional DNA methylation genes which may possible regulate gene expression. Methods:We combinated analysis of methylome and transcriptome of esophageal squamous cell carcinoma using BRB-array Tool and online Venny analysis to identify DNA methylation that may have functional consequences in esophageal squamous cell carcinalma development and progression. We further identified the pathway of these genes using DAVID (Database for Annotation, Visualization, and Integrated Discovery v6.7) online bioinformatics analysis. Results:168genes showed inverse correlation between DNA methylation and expression change.(e.g. decreased expression with increased methylation, and vice-versa). KEGG signaling pathway analysis found that these genes are involved in several cancer-related signaling pathways. Conclusion: Abnormal DNA methylation may affect multiple tumor-associated signaling pathways and these abnormal DNA methylations of genes may be involved in the development and progression of esophageal squamous cell carcinoma. These result suggested that esophageal squamous cell carcinoma is a malignant disease which involved many abnormal genes.The third part:Objective:To detect EPPB41L3, GPX3methylation frequency between tumor tissues and control normal tissues, between esophageal squamous cell carcinoma patients’ plasma and healthy volenteers’ plasma, and to evaluate the diagnostic value of the EPB41L3,GPX3methylation status in the plasma for esophageal squamous cell carcinoma. Methods:We collected esophageal squamous cell carcinoma tissues (n=42cases) and adjacent surrounding normal tissues (n=42cases), plasma of the esophageal squamous cell carcinoma (n=42cases) and plasma of the healthy individuals (n=50cases). We used Methylation specific PCR (MSP) combined with agarose gel electrophoresis to detect the methylation status of the EPB41L3, GPX3. We used SPSS13.0software for statistical analysis by χ2test and Fisher’s exact test. Results:EPB41L3frequency of methylation in tumor tissue was59.5%, significantly higher than the adjacent tissues which was4.8%, the difference was statistically significant (χ2=28.873, P <0.001, n=84). GPX3methylation frequency was54.8%in tumor tissue, adjacent tissues was9.5%, the difference was statistically significant (χ2=19.704, P <0.001, n-84). In the plasma of cancer patients EPB41L3methylation frequency was31.0%, GPX3was40.5%, and the methylation of EPB41L3and GPX3in the plasma of healthy volunteers was not detected. Receiver operating characteristic curve (ROC curve) analysis found that when any one of the EPB41L3and GPX3methylation was positive, the sensitivity rose to57.1%and the specificity is still100%, AUC=0.786(95%CI=0.685-0.886). Conclusion:The methylation frequency of EPB41L3and GPX3is higher in tumor tissus than control normal tissues, in plasma of cancer patients than in plasma of healthy volunteers. Combination detection of the methylation status of EPB41L3and GPX3can improve the sensitivity without compromising the specificity. |
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