| 1 Background and objectiveEsophageal cancer (EC) is one of the six most common malignant tumors. In China, Taihang Mountain areas at the junction Henan, Hebei and Shanxi provinces have been documented as the world's highest incidence and mortality rate of EC. EC has a striking geographic distribution worldwide, with higher prevalence in some areas of China, which may suggest environmental factors play important roles in the occurrence of esophageal cancer. Although tobacco smoking and alcohol drinking account for over 90% of esophageal squamous cell carcinoma (ESCC) in West, different research groups in Asia or China have found that the association with smoking and drinking for ESCC risk is strikingly inconsistent. Therefore, we evaluated the association of smoking, drinking and esophageal cancer by this survey of large sample sizes in high incidence areas, China.Environmental factors can cause the major molecular changes of DNA damage is DNA methylation. Strong environmental factors can cause methylation, while they disappear or other factors affect, it also is back to normal. Mutation, translocation or deletion in gene can also cause methylation of specific sites. A lot of literature has said that abnormal methylation of tumor suppressor genes for the esophageal cancer is often an early molecular event, possibly as early diagnosis of esophageal cancer biomarker. Methylated DNA in peripheral blood is more stable, and therefore abnormal methylation in peripheral blood test has an important value in early diagnosis of cancer. A large number of researches have reported the most often used technique of esophageal cancer in different populations detects methylation frequency of a gene or a few one. Detection of these sites is limited, hardly to find key methylated genes. Therefore, true specific marker using for clinical diagnosis in the future, must use high-throughput technology, searching candidate methylation sites of esophageal cancer.However, as compared the majority of cases and controls, the traditional methylated markers of malignancy and the identification are determined. These people are mostly sporadic individuals, from the genetic point of view, before developed the tumor, there are many differences between them, most of which may be purely individual differences and be nothing to do with the tumor; when adult, a part of the group becomes patients with tumors, during this period, emergence of new tumor-associated genetic changes, but at this time of the controls, this differences in tumor-related genes and the individual differences are mixed, for screening and identification of the true tumor-associated methylated gene brings great distress. How to reduce or narrow this context differences, is the screening of esophageal cancer-specific methylation markers encounters the most challenging problem.In this study, a reversal of the previous search from different tumor markers, using unique esophageal cancer patients with the monozygotic twins as subjects, molecular differences between case and control, compared with two members from discordant tumor-twins can determine tumor-associated methylated genes. Since in theory the genetic background between twins is identical when birthed, which reduces the maximum individual differences caused by the "noise" genes, as provides a unique way, for we may get specific tumor markers in esophageal cancer.We used three high throughput technologies to detect critical methylation sites in monozygotic twins with esophageal squamous cell carcinoma, looking for the development of esophageal cancer candidate methylated genes, to provide a theoretical basis clarifying the roles of esophageal cancer, and further provide a candidate target for high-risk subjects screening and early diagnosis.2 Materials and methods2.1 Subjects7548 cases from high incidence areas of esophageal cancer were recruited from Anyang Tumor Hospital, Linzhou Tumor Hospital, Heping Hospital (the hospital affiliated with Changzhi Medical College) and Cixian People Hospital at the junction of Henan, Hebei and Shanxi provinces. The majority of patients were pathologically confirmed esophageal squamous cell carcinoma after 2006 year. Mainly after 2009 13014 controls were the above-mentioned hospitals'out-patients and population volunteered to participate in endoscopic screening, excluded esophageal and gastric cancer by endoscopic examination.2 male discordant EC twin-pairs were collected from the high incidence areas of esophageal cancer in Henan. They all were extracted from 3ml blood samples and esophageal mucosal lesions were confirmed by histopathologic examination of biopsy.2.2 Questionnaires and interviewsWe collected information about cases and controls from above-mentioned areas, moreover, we took face-to-face interviews for twins and patients (over 80%), and conducted Questionnaires surveys, including personal circumstances, educational level, smoking and drinking alcohol history, family history of cancer, living habits and family income, etc. Finally, it is very important to obtain clinical data about the patients'further details in the hospitals.2.3 Zygosity diagnosis of twinsWe confirmed by similarity, ABO blood type and short tandem repeat.2.4 Human Methylation Bead Chip HybridizationWe used including 27578 CpG sites Illumina Infinium Human Methylation27 Bead Chip to detect ESCC methylation changes in 2 twin pairs who are discordant for ESCC. We extracted DNA using Qiagen blood kit, after quality controls, converted C into T in CPG islands which didn't change the DNA methylation, then and amplified DNA, digestion, precipitation and suspension, chip hybridization, Washing, dyeing and protection, scanning.2.5 Statistical analyses2.5.1 Analysis of large-scale DataAll the collected clinical data were inputted in excel sheets, using SPSS 17.0 statistical software, taking theχ2 test,α=0.05 test level of bilateral and multi-factors logistic regression analysis (Penter=0.05, Premoved=0.10).2.5.2 Analysis of methylating DataWe used Bead Studio's Methylation Module v3.2 software for data analysis, and according to P value<0.004 and absolute value of Delta Beta≥0.17 screened different methylation sites.3 Results3.1 The association with environmental factors and risk for esophageal cancerAfter adjusting gender and age, smokers had a 1.17 fold higher risk for esophageal cancer than non-smokers; while no association was found with alcohol drinking.3.2 The association with DNA methylation and esophageal cancer in MZ twinsVia DNA different methylation sites for ESCC and controls in MZ twins, we found that 2 twin pairs shared 10 significantly different methylation sites, which each corresponded to 10 methylated genes such as KCNE1, TP53I3, NFE2, TNFSF13B, AIM2, CTAGE5,ZNF185,ARID3A, OR10J1 and HIPK3.4 Conclusions4.1 Large-scale epidemiological surveys on patients with esophageal cancer was foundSmoking is the high risk factors for esophageal cancer in high-risk areas, China.4.2 Analysis on DNA methylation of discordant EC in MZ twins We found that 10 significantly different methylated genes in the ESCC and normal blood, which may easily search key methylated genes with esophageal cancer from a variety of ways. |
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