A DNA Methylation Prognostic Signature Of Lung Adenocarcinoma And The Biological Function Study

Objectives?1?To find the methylation CpG sites associated with the prognosis of lung adenocarcinoma patients with the information of genome-wide methylation microarrays in TCGA?The Cancer Genome Atlas?database.?2?To validate the effect of DNA methylation prognostic signature found in TCGA database in lung adenocarcinoma patients of Chinese population and to evaluate the prognosis prediction efficiency of prediction model.?3?To explore the influence of different methylation level on the growth and the expression of gene and protein in lung adenocarcinoma cell lines,further to study the molecular biology mechanism of methylation on the development of lung adenocarcinoma.Methods?1?The screening of methylation CpG sites associated with the prognosis of lung adenocarcinoma based on TCGA:The omics data of lung adenocarcinoma in TCGA database were used in this study,including methylation,mRNA expression and clinical information.Paired t test was used to evaluate the differential methylation CpG sites between tumor tissue and adjacent tissue,then LASSO COX regression was preformed to select the methylation CpG sites which was the associated with the prognosis of lung adenocarcinoma.The prognosis prediction efficiency of prediction model was estimated with the value of Harrell's C-statistic.The correlation between methylation level and gene expression level was analyzed using a linear correlation model.?2?The effect of methylation CpG sites on the prognosis of lung adenocarcinoma in Chinese:The subjects of this study were collected in Wuhan Tongji Hospital in 2014.The subjects were all diagnosed with lung adenocarcinoma and an operative treatment.The tumor tissue and adjacent tissue of patients who was contacted in the following were collected.Pyrosequencing was used to test the methylation level of the methylation CpG sites in tumor tissue and adjacent tissue.COX regression model was performed to evaluate the association between methytion signature and the prognosis of lung adenocarcinoma.The prognosis prediction efficiency of prediction model was estimated with the value of C-statistic.The relationship between methytion signature and clinical characteristics was also assessed.RT-PCR and Western blot were taken to detect the gene and protein expression level.The correlation between methylation level and gene expression level was analyzed using a linear correlation model.?3?The biological function study of methylation CpG sites in lung adenocarcinoma cell lines:Human lung adenocarcinoma A549 cells,NCI-H1975 and immortalized human bronchial epithelia MRC-5 cells were cultured in vitro and demethylated with5-aza-2dC?5-aza-2'-deoxycitydine?in different concentration?0?mol/L,1?mol/L,5?mol/L,10?mol/L?.MTT was performed to detect the state of cell growth.Pyrosequencing was used to test the methylation level of cell lines with 5-aza-2dC treatment.RT-PCR and Western blot were taken to detect the gene and protein expression level in Human lung adenocarcinoma cell lines and normal cell line.Results?1?The screening of methylation CpG sites associated with the prognosis of lung adenocarcinoma based on TCGA:There were 418 patients who had the information of both TCGA methylation array and clinical data.14,9486 methylation CpG sites were with a FDR<0.05 in the comparison between tumor tissue methylation level and adjacent tissue methylation level and 1291 methylation CpG sites had an absolute value of log₂?fold change?more than 2.5.With LASSO COX regression analysis,three methylation CpG sites were selected which was the associated with the prognosis of lung adenocarcinoma?cg14517217,cg15386964 and cg18878992?.When estimating the association between methytion signature and the prognosis of lung adenocarcinoma Multivariate COX regression model revealed that methytion signature was associated with increased death risk for lung adenocarcinoma patients?HR=45.30,95%CI=6.69-306.83;P<0.001?.When the subjects were divided into high-risk and low-risk groups based on the median of methylation signature,the high-risk group people had a 123%higher death risk than low risk group people?HR=2.23,95%CI=1.41-3.54;P<0.001?.The C-statistic value was elevated to 0.77 when added age,gender,race,and other clinical variables into signature to make a prediction model,compared to using signature alone?C-statistic:0.66?.In addition,we found that the mRNA expression level of SEPT9 and HIST1H2BH was higher in tumor tissue than in adjacent tissue,while lower in MAPT?P<0.05?.The correlation coefficient between methylation level and gene expression level of these three methylation CpG sites were0.32,0.71 and-0.35,significantly?all P values<0.001?.?2?The effect of methylation CpG sites on the prognosis of lung adenocarcinoma in Chinese:This study included 116 lung adenocarcinoma patients with success following-up.When estimating the association between methytion signature and the prognosis of lung adenocarcinoma,the methytion signature was associated with increased death risk for lung adenocarcinoma patients?HR=8.81,95%CI:2.72-28.51;P<0.001?.Compared with low risk group people,the death risk of high-risk group people was 7.52 times higher?HR=8.52,95%CI:0.52-138.58;P=0.132?.The C-statistic value of prediction model was elevated to 0.73 compared with the signature alone?C-statistic:0.61?.The methyaltion level was higher in tumor tissue compared with adjacent tissue in these three methylation CpG sites in 30 lung adenocarcinoma patients?P<0.05?.In addition,we found that the mRNA expression level of SEPT9 and HIST1H2BH was higher in tumor tissue than in adjacent tissue in 30 lung adenocarcinoma patients,while lower in MAPT?P<0.05?.The correlation coefficient between methylation level and gene expression level of these three methylation CpG sites were 0.31?P=0.095??0.41?P=0.024?and-0.28?P=0.138?.?3?The biological function study of methylation CpG sites in lung adenocarcinoma cell lines:The growth of cell lines was inhibited when the cells were demethylated with5-aza-2dC,especially in A549 and NCI-H1975.In control groups?without treatment?,the MRC-5 cell line was showed slightly methylated,while A549 and NCI-H1975 were all highly methylated.The effect of 5-aza-2dC treatment seemed not obvious on MRC-5cell line while the methylation level was decreased with 5-aza-2dC treatment in A549and NCI-H1975 cell lines.5-aza-2dC treatment presented no obviously influence on the mRNA expression level as well as protein expression level on MRC-5 cell line.For A549 and NCI-H1975,the mRNA and protein expression level of SEPT9 and HIST1H2BH?cg14517217 and cg15386964?was down-regulated with different concentration of 5-aza-2dC treatment,the mRNA and protein expression level of MAPT?cg18878992?was up-regulated with different concentration of 5-aza-2dC treatment.Conclusions?1?With the TCGA data analysis in lung adenocarcinoma,we have found a DNA methyaltion prognosis signature based on three methylation CpG sites which were associated with the prognosis of lung adenocarcinoma?cg14517217,cg15386964 and cg18878992?.In addition,the prognosis prediction efficiency of prediction model was satisfactory in lung adenocarcinoma patients of Chinese population.?2?With the exploration on the biology mechanism of methylation on lung adenocarcinoma in human lung adenocarcinoma,MAPT may be a potential tumor suppressor gene while SEPT9 and HIST1H2BH may be potential oncogenes.The change of methylation level could have an effect on mRNA and protein expression.The biology mechanism of methylation on the development of lung adenocarcinoma may through the causal relationship between methylation and gene/protein expression.