Variable DNA methylation of aging-related genes is associated with male COPD

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a chronic lung inflammatory disease which has a close relationship with aging. Genome-wide analysis reveals that DNA methylation markers vary obviously with age. DNA methylation variations in peripheral blood have the potential to be biomarkers for COPD. However, the specific DNA methylation of aging-related genes in the peripheral blood of COPD patients remains largely unknown.

Methods: Firstly, 9 aging-related differentially expressed genes (DEGs) in COPD patients were screened out from the 25 aging-related genes profile through a comprehensive screening strategy. Secondly, qPCR and multiple targeted bisulfite enrichment sequencing (MethTarget) were used to detect the mRNA level and DNA methylation level of the 9 differentially expressed genes in the peripheral blood of 60 control subjects and 45 COPD patients. The candidate functional CpG sites were selected on the basis of the regulation ability of the target gene expression. Thirdly, the correlation was evaluated between the DNA methylation level of the key CpG sites and the clinical parameters of COPD patients, including forced expiratory volume in one second (FEV1), forced expiratory volume in one second as percentage of predicted volume (FEV1%), forced expiratory volume/ forced vital capacity (FEV/FVC), modified British medical research council (mMRC) score, acute exacerbation frequency and the situation of frequent of acute aggravation (CAT) score. Lastly, differentially methylated CpG sites unrelated to smoking were also determined in COPD patients.

Results: Of the 9 differentially expressed aging-related genes, the mRNA expression of 8 genes were detected to be significantly down-regulated in COPD group, compared with control group. Meanwhile, the methylated level of all aging-related genes was changed in COPD group containing 219 COPD-related CpG sites in total. Notably, 27 CpG sites of FOXO3 gene showed a lower False Discovery Rate (FDR) and higher methylation difference values. Also, some variable DNA methylation is associated with the severity of COPD. Additionally, of the 219 COPD-related CpG sites, 147 CpG sites were not related to smoking.

Conclusion: These results identified that the mRNA expression and DNA methylation level of aging-related genes were changed in male COPD patients, which provides a molecular link between aging and COPD. The identified CpG markers are associated with the severity of COPD and provide new insights into the prediction and identification of COPD.

Keywords: Aging; Aging-related genes; COPD; DNA methylation.

Fig. 1

The mRNA level of the aging-associated genes FOXO3, TP53, TGFβ1, MMP2, HDAC1, NUF2, ATG3, and AREG were significantly down-regulated in peripheral blood of COPD patients. (a-h) The mRNA expression of these genes was detected by qPCR. *** p < 0.001; **** p < 0.0001

Fig. 2

Volcano plot of differential methylation CpG sites between COPD patients and normal volunteers. The up-regulated sites were presented as red dots and down-regulated as green. p-value< 0.05

Fig. 3

Correlation analysis between differential methylation sites and other clinical indicators. Correlation analysis between differential methylation sites and clinical indicators. a. CpG sites positively associated with the mMRC score (0–4). p-value< 0.05. The methylation differences at 23 sites between different mMRC groups are presented in the clustering map. b ROC curve of acute exacerbation frequency p-value < 0.05, AUR > 0.5. c ROC curve of CAT score. p-value < 0.05, AUR > 0.5

Fig. 4

Venn diagram of the intersection of aging-related CpG sites and smoking-related CpG sites