A functional methylation signature to predict the prognosis of Chinese lung adenocarcinoma based on TCGA

Abstract

Background: Lung cancer is the leading cause of cancer morbidity and mortality worldwide, however, the individualized treatment is still unsatisfactory. DNA methylation can affect gene regulation and may be one of the most valuable biomarkers in predicting the prognosis of lung adenocarcinoma. This study was aimed to identify methylation CpG sites that may be used to predict lung adenocarcinoma prognosis.

Methods: The Cancer Genome Atlas (TCGA) database was used to detect methylation CpG sites associated with lung adenocarcinoma prognosis and construct a methylation signature model. Then, a Chinese cohort was carried out to estimate the association between methylation and lung adenocarcinoma prognosis. Biological function studies, including demethylation treatment, cell proliferative capacity, and gene expression changes in lung adenocarcinoma cell lines, were further performed.

Results: In the TCGA set, three methylation CpG sites were selected that were associated with lung adenocarcinoma prognosis (cg14517217, cg15386964, and cg18878992). The risk of mortality was increased in lung adenocarcinoma patients with the gradual increase level of methylation signature based on three methylation sites levels (HR = 45.30, 95% CI = 26.69-66.83; p < 0.001). The C-statistic value increased to 0.77 when age, gender, and other clinical variables were added to the signature to prediction model. A similar situation was confirmed in Chinese lung adenocarcinoma cohort. In the biological function studies, the proliferative capacity of cell lines was inhibited when the cells were demethylated with 5-aza-2'-deoxycytidine (5-aza-2dC). The mRNA and protein expression levels of SEPT9 and HIST1H2BH (cg14517217 and cg15386964) were downregulated with different concentrations of 5-aza-2dC treatment, while cg18878992 showed the opposite result.

Conclusion: This study is the first to develop a three-CpG-based model for lung adenocarcinoma, which is a practical and useful tool for prognostic prediction that has been validated in a Chinese population.

Keywords: DNA methylation; biological mechanism; lung adenocarcinoma; prediction model; prognosis.

FIGURE 1

Flow chart of the study design. Twenty‐six paired tumor and adjacent normal samples of lung adenocarcinoma patients were used for candidate methylation CpG islands screening with Illumina Human Methylation 450 platform in TCGA set. Moreover, LASSO Cox regression method was used to build a methylation signature model in the validation set. In addition, the three‐CpG‐based prognostic signature was validated in a Chinese dataset. Biological function study of methylation CpG sites in lung adenocarcinoma cell lines was further performed

FIGURE 2

Bioinformatics analysis based on the TCGA set. (A) Heatmap showing the top 100 differentially methylation CpGs in 26 paired tumor and adjacent normal tissues of lung adenocarcinoma. (B) GO enrichment analysis using the top 100 differentially methylation CpGs in 26 paired samples. (C) and (D) LASSO Cox regression analysis based on the TCGA set. (C) LASSO coefficient information of candidate methylation CpG islands. (D) Two dotted vertical lines were drawn in the optimal criteria of 1‐s.e. and with a result of three nonzero coefficients. Three CpGs, cg14517217, cg15386964, and cg18878992, with coefficients of 0.45, 0.04, and −0.17, were eventually incorporated into the model

FIGURE 3

The influence of 5‐aza‐2dC on the methylation level in different cell lines. Human lung adenocarcinoma A549 cells, NCI‐H1975 cells, and immortalized human bronchial epithelia MRC‐5 cells were cultured in vitro. Different concentrations of 5‐aza‐2dC were added to these three cell lines: 0 μmol/L (control group), 1 μmol/L (low‐demethylated group), 5 μmol/L (middle‐demethylated group), and 10 μmol/L (high‐demethylated group). Pyrosequencing was conducted to validate the CpG methylation levels

FIGURE 4

Cell growth analysis via the MTT assay. A549, NCI‐H1975, and MRC‐5 cells were demethylated with 5‐aza‐2dC at different concentrations (0 μmol/L, 1 μmol/L, 5 μmol/L, and 10 μmol/L). MTT was performed to evaluate cell growth for 6 days. *p < 0.05

FIGURE 5

The gene expression of three methylation CpG islands in three cell lines by RT‐PCR. (A) RT‐PCR showing the gene expression of three methylation CpG islands in cells demethylated with 5‐aza‐2dC at different concentrations. (B) The gene expression of three methylation CpG islands in cells demethylated with 5‐aza‐2dC. **p < 0.001