Study On Aberrant CpG Island Methylation Pattern Of Tumor-related Gene Promoter Induced By Folate Deficiency And Its Role In Tumorigenesis Of Sporadic Colorectal Cancer

Colorectal cancer (CRC) is the most common gastrointestinal tract malignancy. Globally, the majority of cases occur in developed countries, particularly North America, Australia and Western Europe. Observational studies comes to evidence that causes of CRC are largely environmental compared to age or sex matched controls and the main characteristic in these studies has been a change from a prudent diet to a Westernized diet with higher intake of energy dense foods and lowered physical activity. Great advances in epigenetic modifications have been made in understanding these ecological findings. Epigenetic processes are essential in normal development and differentiation but may be misdirected and predispose to tumorigenesis. The most well-known epigenetic marker remains to be DNA methylation. Distinguished from genetic changes, epigenetic changes are potentially reversible which would provide promising targets for preventive and therapeutic interventions. Diet is the major aspect of the environment may influence DNA methylation and especially interesting are nutrients, which are needed for nucleic acid and DNA synthesis and for the enzymes regulating their syntheses, for example, essential amino acids, zinc, folate, and vitamins.One of most strongly implicated nutrients is folate, which is a generic term for a naturally occurring family of B-group vitamins and a natural constituent of foods such as green leafy vegetables, legumes, and citrus fruits. An inverse relationship between folate intake and risk of CRC was also demonstrated in follow-Up studies. Folate through its role in the one carbon metabolism is crucial for both DNA synthesis as well as methylation. The effects of folate deficiency on DNA methylation patterns has been studied, however, results from studies are inconsistent, with some showing hypomethylation, no change or hypermethylation and the validity of measuring methylation patterns in CRC as a surrogate marker of methylation still remains unknown.In this study, we investigated the methylation patterns changes induced by folate deficiency in normal human colon mucosal epithelial cell line, NCM460 cultured with varying folate concentrations with a high-resolution mapping assay:NimbleGen Methylated DNA Immunoprecipitation (MeDIP) chip assay. Aberrant methylation pattern in promoter of related genes were confirmed in tumor tissues and corresponding nonmalignant tissues by bisulfate sequencing PCR (BSP). There were 17 genes with hyper or hypomethylation in the CpG island of promoter on chromosome 21 and 8 of them seemed to be association with tumorigenesis of CRC. The association between the promoter methylation patterns of cystathionine-beta-synthase (CBS) which is involved in methyl metabolism and its expression level was detected by real-time RT-PCR and immunostaining. At the same time, the functional meaning of CBS promoter hypermethylation was also analyzed by detecting its direct substrate, homocysteine (HCY). The data of CBS promoter methylation level statistically correlated with pathological parameters and clinical outcome. Univariate analysis showed CBS hypermethylation level correlated with tumor stage, recurrence, cancer related death, recurrence-free probability and overall survival. Multivariate analysis showed CBS hypermethylation level correlated with recurrence rate and overall survival independent of tumor stage. Our study disclosed that folate deficiency could induce aberrant genes methylation and expression in CRC. Some related genes play roles in tumorigenesis and would serve as prognostic markers for tumor progression.