Association Studies Of Promoter Hypermethylation Of Multiple Candidate Genes With Lung Cancer

Objectives: Lung cancer is the most deadly cancer around the world.Although the treatments and diagnosis are improved,the five-year survival rate for lung cancer is still very low.Promoter CpG island methylation is an important mechanism for the regulation of gene expressions.Promoter methylation of oncogenes or tumor suppressor genes can affect biological behaviors of tumors.On the other hand,new biomarkers for tumor diagnosis might be discovered through data analysis of promoter methylations of tumor-related genes.Candidate genes were selected through database screening and then analyzed and verified the situations of promoter methylation.We hope to provide new experimental data to understand the pathological mechanism of lung cancer and discover potential biomarkers for lung cancer diagnosis.Methods: Promoter methylations of lung cancer-related genes in NSCLC tissue,para-cancer tissues and remote para-cancer tissues were measured by quantitative methylation specific PCR(qMSP).The relevant positive gene promoter methylation was analyzed in combination with the data from TCGA database,using methylation reference percentage(PMR)to represent methylation level.The non-parametric Wilcoxon test was used to compare the methylation levels among the three groups of samples.Finally,relevant cell function verification was carried out for the three positive genes screened.Promoter activity was measured by the dual luciferase assay.Lung cancer cell lines were treated with 5'-aza-deoxycytidineand the expressions of TNFRSF10 C,PRKCDBP and EPAS1 were measured by qPCR.Results: 1.Twenty two genes were selected for promoter methylation measurements after primer verification.2.Promoter methylation levels of these 22 genes were determined and statistically analyzed.Three genes(TNFRSF10C,PRKCDBP and EPAS1)were selected.Comparison of the methylation levels of tumor and the para-tumor showed the promoter methylation levels of PRKCDBP and EPAS1 had diagnostic significance for lung cancer.The methylation level of TNFRSF10 C was also higher in NSCLC tumor samples than that in the para-tumors(median PMR: 2.73%vs.0.75%,P = 0.013).The methylation level of PRKCDBP was higher in tumor than that of the para-tumor(median PMR,1.46%vs.0.42%,P = 0.019).PRKCDBP methylation level had diagnostic significance for lung cancer(AUC=0.717,P = 0.0005,95% CI =0.609-0.824,sensitivity=59.09%,specificity=77.27%,P = 0.0003).The ROC curve(AUC=0.722 P = 7.955E-4,95%ci =0.606-0.838,sensitivity=40%,specificity=97.30%)was plotted by calculating the methylation level of EPAS1 in the tumor and the para-tumor.It was found that the methylation level of EPAS1 had diagnostic significance for lung cancer.3.According to the TCGA database of lung cancer in cBio,the methylation levels of TNFRSF10C(r =-0.379,P = 0.008),PRKCDBP(r =-0.437,P = 4.88E-10)and EPAS1(r =-0.504,P = 3.5E-13)were negatively correlated with the expression of mRNA.4.Lung carcinoma cell lines A549 and H1299 were demethylated with 5 '-aza-deoxycytidine,and then the mRNA expressions of TNFRSF10C(A549: Fold change(FC)= 8,P = 0.0001),PRKCDBP(A549: P=0.003,FC=2.193;H1299: P=5.713E-7,FC=32.081),EPAS1(A549: P = 3.0516-5,FC=3.346;H1299: P = 6.E-289 7,FC = 3.267)were determined by qPCR.5.Dual luciferase assay showed that the transcriptional activity of the three promoter regions of the target gene fragments was significantly higher than that of the control group.The promoter regions of TNFRSF10C(FC = 1.570,P = 0.011),PRKCDBP(P = 0.002,FC = 9.54),EPAS1(P = 0.044,FC = 2.427)could regulate transcription.Conclusion: The methylation levels of TNFRSF10 C,PRKCDBP and EPAS1 promoter were different in lung cancer tumor,para-tumor and distance-non tumor samples,and had statistical significance.Further analysis showed that all three genes were correlated with the occurrence and development of lung cancer.These results suggested that TNFRSF10 C,PRKCDBP and EPAS1 could be used as potential markers for lung cancer diagnosis.