Detection And Identification Of Abnormal DNA Methylation Pattern In Gene Promoter And Investigation Of The Relationship Between The DNA Methylation And Mutation In Early Non Small Cell Lung Cancer

Lung cancer,also known as lung carcinoma,is one of the world's highest incidences of malignant tumors.Early detection and early treatment is the key to fight against lung cancer.DNA methylation is one of the events in the early stage of lung cancer.These changes in DNA methylation can serve as a potential biomarker in early detection of lung cancer and then improve survival rates in lung cancer patients.Meanwhile,DNA methylation abnormality has a significant impact on the incidence of lung cancer,as well as the development and the prognosis and drug resistance.Therefore,studies of abnormal DNA methylation pattern of early stage lung cancer may be helpful for studying of molecular mechanisms and identification of novel biomarkers for diagnosis and prognosis prediction.With the development of high-throughput sequencing technologies in recent years,we have the opportunity to accurately detect DNA methylation changes in whole genome level.This study performed a simplified reduced representation of bisulfite sequencing(RRBS)high-throughput analysis to identify early stage lung cancer abnormal DNA methylation in tissue samples.Firstly,4775 differentially methylated CGs was found between paired cancer tissues and adjacent tissues.Based on the clustering analysis,we found that llung cancer samples and adjacent samples are clustered in two groups,which indicated that there might be a consistent methylation pattern in the early stage lung cancer samples.Hypomethylated-CGs were more than hypermethylated-CGs,which was also consistent with the reality that cancer cells had a globally decreased methylation level and hundreds of specific hypermethylated sites.The annotation results showed that hypermethylated-CGs in the promoter region accounted for 24%of all hypermethylated-CGs,while only 13.16%hypomethylated-CG was found in promoter region.These results indicated that the hypermethylation of CGs was concentrated in the promoter regions with CpG Island.Therefore,DMR(Differentially Methylated Region)analyses were performed to further investigate the methylation pattern within promoter regions.After DMR analysis,9 highly methylated DMRs were found in six lung cancer samples including PTTG1IP,DRICH1 and AC139103.1 promoter regions.We further verified the abnormal promoter methylation patterns of PTTG1IP promoter in more lung cancer tissues and cell lines,as well as the data from TCGA.PTTG1IP promoter methylation level was negatively associated with the low mRNA expression levels in tumor tissues and in cell lines.The expression of PTTG1IP was significantly up-regulated by decreasing promoter methylation level by using 5-aza-dC.The cell proliferation results showed that overexpression of PTTG1IP in lung cancer cell line AS49 could inhibit cell proliferation.Taken together,we hypothesized that PTTG1IP was a was tumor suppressor gene in early lung cancer cells.It was hypermethylated in promoter region and as a result,was decreased in mRNA level,then reduced the inhibition of proliferation of lung cancer cells.Besides,KEGG and GO analysis revealed that the abnormal regions of methylation pattern were mainly concentrated in mTOR and MAPK signal pathways,which were related to cell proliferation,differentiation and apoptosis.These results suggested that some of these pathways have been abnormal in early stage lung cancer.Secondly,we all knew that both genetic and epigenetic alterations were involved in the development and progression of cancer.In order to further elucidate the role of mutation and DNA methylation modification in lung cancer,we performed an analysis to identify mutations from RRBS data.In this study,the bioinformatic screening process of tumor specific mutations(Tumor Specific Variant,TSV)was developed based on single nucleotide variation criterion in bisulfite converted DNA sequencing data.364 multiple mutated(?4)gene promoters were found,which mainly concentrated in Phosphatidylinositol signaling system.We have also identified mutations in known tumor suppressor genes including TP53.To further reveal the general relationship between DNA methylation and mutation,whether mutation was one of the causasive events of abnormal DNA methylation pattern,we analyzed the linkage associations of mutation and DNA methylation level in the areas having TSVs with 40%-60%mutation rate in cancer samples.From the results,we found that both A->G and T->C mutations might cause more increase of fluctuation of DNA methylation level of nearby area.In addition,we also found that the methylation levels of CGs close to TSVs were significantly lower in mutated reads.X->A(X:T,G,C)and X->T(X:A,G,C)mutations reduced methylation levels of nearby CGs to 7%relatively.In conclusion,the crosstalk between TSVs and methylation levels was suggested in our study.Further investigation was required to reveal the mechanisms underlying this interaction during the occurrence and development of lung cancer.