Genome-wide DNA Methylation Profile In Esophagus Squamous Cell Carcinoma

Background and Objective:Aberrant methylation of CG islands in the gene promoters can result in the inhibition of gene transcription, decrease of protein expression, and loss of the gene function. Genome-wide hypomethylation and aberrant hypermethylation in some specific genes are usually observed when human cancer occurs. Previous studies have reported the relationship between the promoter methylation of tumor-suppressor genes and the occurrence, development of tumors such as esophageal cancer. As DNA methylation pattern has tumor specificity, different types of cancer may have different DNA methylation spectrum. Therefore, we assume that identification of abnormal methylation panel and construction of specific profiles of specific genes may be potential biomarkers for the diagnosis and prognosis of cancer.Traditional methods for analyzing the DNA methylation require tissue samples, which limit its clinical application. Free circulating DNA, a cellular extracellular DNA widely existing in plasma and serum of cancer patients, have consistent genetic characteristics and methylation changes with tumor cells, which can be an alternative biomarker for disease diagnosis, treatment outcome evaluation and prognosis prediction. This study aims to compare the consistency of DNA methylation between esophageal cancer tissues and cell free DNA extracted from the same patient’s plasma, construct DNA methylation profiles of cancer suppressor genes, and explore the feasibility of applying cell free DNA based methylation profile as a biomarker for early diagnosis of esophageal cancer.Methods:We used Illumina450K bead-chip to detect methylation status in esophageal squamous cell cancer tissues,remote’normal’ esophageal tissues from the same patient and normal esophageal mucosa samples from healthy controls. Potential genes were filtrated according to delta beta value and diffscore.Based on the top300CG sites with extreme P values incombination with GO (Gene Ontology) function, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, Genecards database and the literature reports,10genes were selected for the subsequent verification. By using the mass spectrometry technology, we quantified the methylation status of DNA from esophageal squamous cell carcinoma tissues and paired plasma. The consistency of DNA methylation between these two types of samples was tested.Results:Cancer tissues and adjacent esophageal tissues had similar methylation patterns which were obviously different from normal tissues. Hypermethylation sites were mainly distributed in the CG island and promoter region, while hypomethylation sites were mainly distributed in the non-CG island and the non-promoter region. Considering the biological functions and literature reports, we constructed a panel of aberrant methylation biomarkers, which included10tumor suppressor genes (ADAMTS9, AIM2, CASZ1, CDH13, EBF3, ING2, IQGAP2, KLF6, TMEFF2and TRIT1). We used the flight mass spectrometry to validate38CG sites from these10genes.The methylation level was higher in the samples from plasma, followed by carcinoma tissues and normal tissues. The methylation of cell free DNA extracted from the plasma had higher consistence with the DNA originated from the cancer tissues,which indicated that cell free DNA from the plasma could be an alternative biomarker for the diagnosis of esophageal cancer.Conclusion:Gene-chip can be applied for the preliminary screening for differential methylated genes. Aberrant methylation panel of genes in esophageal cancer should be validated before being applied as a clinical biomarker. Plasma cell free DNA can be applied as a potential biomarker for the diagnosis of esophageal cancer.