Methylation Analysis In Lung Cancer Based On High-density Genomic DNA Methylation Chips Of Xuanwei And Fuyuan And Methylation Level Validation Of Differentially Genes

Lung cancer,also known as bronchial cancer,is the most common malignancies in the world.The morbidity and mortality rate rank the highest among the malignancies of China.The lung cancer mortality rate of Xuanwei City and Fuyuan County of the Yunnan Province is much higher than other areas in China,moreover the incidence and mortality in females are higher than in males in some regions.Lung carcinogenesis is related to environmental factors.It has been proposed that DNA methylation plays an important role in the development of lung cancer.In order to explore the pathogenesis of lung cancer in Xuanwei and Fuyuan areas and the influence of environmental carcinogens on DNA methylation,we first analyze the genome-wide DNA methylation level in both Xuanwei and Fuyuan lung cancer tissues and cell lines,and then further evaluate the influence of BaP treatment on 16HBE and HBE-pic cell lines methylation level.We applied a genome-wide methylation DNA immunoprecipitation microarray chips(MeDIP-chip)to investigate the methylation profile difference between Xuanwei&Fuyuan lung cancer tissues and peritumorial tissues(n=12 cases);the genome-wide methylation level of Xuanwei lung cancer cell lines and BaP treated 16HBE and HBE-pic cell lines were detected using Infinium human methylation 450k beadchip.The result reveals that there were 54183 differentially methylated sites in cancer tissues in comparison with peritumorial tissues,34004 hypermethylation genes;18814 genes in the promoter regions were abnormally methylated,and 11539 of those genes were hypermethylated.BaP treatd 16HBE and Xuanwei lung cancer cell lines share a lot of differentially methylation sites.In the study,many novel lung cancer related genes are found to have promoter CpGs hypermethylation,through the combined analysis of lung cancer tissues and cell lines methylation microarray results.And then,we selected gene DKK2、EN1,with promoter region hypermethylation and gene LPAR2,with promoter region hypomethylation,to carry out further methylation and mRNA expression level analysis,in 12 cases of lung cancer tissue compared with peritumorial tissue.We found that:DKK2 and En1 which showed hypermethylation,were also associated with mRNA expression level downregulation,in 10 cases and 12 cases of lung cancer respectively;LPAR2 were hypomethylated in 3 cases of lung cancer tissue compared with peritumorial tissue,and the corresponding mRNA expression level were up-regulated;In 16HBE cell lines,BAP could lead to DKK2 and Enl gene promoter regions CpGs methylation level increase and LPAR2 gene promoter regions GpGs methylation leve decrease.Our research results indicates that BaP can cause genome and gene specific abnormal methylation by epigenetic pathways.Environmental carcinogen-induced genome abnormal methylation is one of the mechanisms of lung cancer carcinogenesis in Xuanwei&Fuyuan.DKK2 and Enl and LPAR2 in Xuanwei&Fuyuan lung cancer tissue samples were abnormally methylated,However,the effects of these genes on cell behavior change need further study,by expanding the sample size for more verification.DKK2、Enl and LPAR2 gene promoter region methylation change can be considered as a potential biomarker for early diagnosis of lung cancer in Xuanwei&Fuyuan area.