Screening And Validation Of Colorectal Cancer Related Aberrant Methylation By Genome-wide Methylation Beadchip

Background and ObjectivesColorectal cancer(CRC)is one of the most commonly diagnosed malignant tumors worldwide.About 1.8 million new cases and 0.86 million deaths were reported in 2018.China is one of the countries with low risk of CRC in last century.However,the incidence of CRC increase dramatically along with the development of social economy and the change of traditional diet structure and lifestyle.Therefore,it has become an important public health problem in China.The etiology of CRC is thought to be influenced by environmental risk factors interacting with genetic variability and epigenetic modifications.Genetic determinants have been evaluated extensively,while the epigenetic maintainers remain unclear.DNA methylation is a general epigenetic mechanism in human genome.By regulating the transcription level of functional genes,it has been proved to participate in many important biological processes,such as embryo development,imprinting inactivation,cell differentiation and aging.To date,the epigenome wide association studies(EWASs)have successfully revealed many aberrant methylated promoters in CRC-related genes.Notably,a universal phenomenon that these methylation changes occurred in both precancerous and cancerous lesions was observed.However,little studies have described accurately the evolution pattern of methylation events during the process of malignant transformation of intestinal epithelial cells in colorectal carcinogenesis.Materials and MethodsA three-phase desigin was adopted in this study.In the screening phase,12 colorectal cancer tissues and paired normal tissues were selected for the genome-wide methylation scanning.Candidate Cp G sites were selected based upon cross-validation analyses from the Cancer Genome Atlas(TCGA)dataset.The methylation level of candidate genes was detected by pyrosequencing in the training dataset(lesion tissues and paired normal tissues from 46 CRCs)and the validation dataset(lesion tissues and paired normal tissues from 13 hyperplastic polyps,129 colorectal adenomas and 256 CRCs).Linear mixedeffects model was used to evaluate the methylation changes of selected genes during the process of colorectal neoplasia.ResultsA total of 174,006 aberrantly methylated Cp G sites were identified in the screening phase,of which 22,232 were found to be hypermethylated and 151,774 were found to be hypomethylated.By annotating Cp G sites according to their relative positions to genes and Cp G islands,we found that hypermethylation mostly occurred in gene poromoters with an overlap of Cp G islands,while hypomethylation incline to be far away from functional regions.Cross-validation analyses based on TCGA confirmed 316 promoter methylation changes coupling with irregulated gene expression.Among which,265 were hypermethylated promoters with downregulated gene expression,51 were hypomethylated promoters with upregulated gene expression.Top 8 methylated genes selected for further analyses were as follows: MPPED2,IKZF1,RSPO3,GALR1,COL23A1,EPHA6,INHBA and HKDC1.In training phase,DNA methylation status of MPPED2、IKZF1、RSPO3、GALR1、COL23A1 and EPHA6 were significant hypermethylation in tumor tissues in comparison with paired normal tissues.Hypomethylation of INHBA and HKDC in tumor tissues was observed.In addition,MPPED2,GALR1,INHBA and HKDC1 showed high discriminative performance for lesion discrimination.Therefore,they were included into our validation phase.In validation phase,Significant hypermethylation in MEEPD2 promoter appeared in lesion tissues of hyperplastic polyps,adenomas and CRCs when compared with paired normal tissues.GALR1 was significantly hypermethylated in adenomas and CRCs when compared with paired normal tissue,while no significant methylation change was observed in hyperplastic polyps.The methylation status of INHBA and HKDC1 was significantly decreased since hyperplastic polyps with a gradual motification in adenomas and CRCs.The mixed-effects modeling analyses showed that point estimates of methylation status of MPPED2 and GALR1 increased continuously from normal mucosa,to hyperplastic polyp,to adenoma,and to carcinoma.For both genes,the changing scope was significant in adenoma and carcinoma.The methylation status of INHBA and HKDC1 decreased steadily during the transformation from normal mucosa to carcinoma.The changing scope of methylation status of INHBA was significant since hyperplastic polyp,while HKDC1 was since colonic epithelium adjacent to colorectal cancer.ConclusionsThe current study explored the CRC related methylated genes by genome-wide methylation scanning and public database analyses,and described the methylation changes during the transformation from nomal colonic epithelium to malignant tumor in colorectal carcinogenesis.The main findings are listed as follows:(1)Global methylation changes occurred during the progression of colorectal carcinogenesis: hypermethylation mostly located in gene poromoters with an overlap of Cp G islands,while hypomethylation tend to get far away from functional regions.(2)MPPED2,IKZF1,RSPO3,GALR1,COL23A1 and EPHA6 were hypermethylated genes in CRC.The methylation level of INHBA and HKDC1 was significantly decreased in CRC.The effect of aberrant DNA methylation on GALR1 was more pronounced in colon cancer in comparison with rectal cancer.(3)Hypermethylation of MPPED2 and GALR1 showed cumulative patterns during the progression of colorectal neoplasia.Aberrant hypomethylation of INHBA and HKDC1 occurred sequentially during colorectal neoplastic progression.In general,the hypomethylation events occured earlier than hypermethylation events.