Discussion
Asthma is a common chronic pulmonary disease, and the  incidence  of asthma  has  increased  in the last few decades [27]. With the increased  incidence  of asthma, new preventive strategies and therapies for asthma are urgently needed to further reduce the  morbidity  and  mortality of asthma. Of particular note is the potential causal role of aging in the asthma pathogenesis [28, 29]. Several relevant studies have identified the altered expression of aging-related genes (such as TP53 and FOXO3) in respiratory diseases [30, 31]. The polymorphism of transcription factor FOXO3 was confirmed to regulate the overactivation of mast cells, down-regulation of anti-inflammatory factors and production of cytokines during the pathogenesis of COPD and asthma [32]. FOXO3 deficiency has been confirmed to play an important role in regulating lung inflammation of COPD/emphysema, which has emerged as a new approach to address the development of pulmonary inflammatory diseases [33]. Similarly, TP53 has been implicated in COPD  pathogens is by mediating the senescence of multiple lung cells, and the overexpression of TP53 also could promote the progression of emphysema in COPD patients [31, 34].
Not only that, as a stable epigenetic marker, aging-related CpG sites were either hypo- or hyper-methylated in COPD and other aging-related diseases [35, 36]. Our previous research identified that DNAm was involved in regulating the expression of 9 aging-related genes in peripheral venous blood of COPD patients [18]. As asthma and COPD have similar even overlapping clinical phenotypes in chronic inflammation and decreased lung function. In this study, we further explored the methylation change of the previous screened aging-related genes in peripheral venous blood of asthma patients. Indeed, the association between these screened 9 aging-related genes and asthma have been extensively studied by previous literatures [37-44]. AREG, E2F1, FOXO3,
HDAC1, MMP2, TGFB1 and TP53 have been confirmed as crucial signaling molecules in asthma [30, 45-51]. Although ATG3 is a key central regulator in autophagy induction during aging [52], and NUF2 is closely related closely associated with lung cell senescence [53], their specific role in asthma has rarely been studied. The differential expression of ATG3, FOXO3, NUF2 and TP53 in asthma patients were also aligned with former studies [30, 53-55]. In addition, excessive secretion of AREG in the airway after acute asthma attack promote airway remodeling [51]. However, AREG is downregulated in peripheral blood of elderly asthma patients, which may be attributed to the different  disease  stages. It is particularly worth noting that the decreased expression of E2F1 in asthma patients is consistent with what we have previously observed in COPD patients [18], which is different from that in lung cancer patients [55]. One possible reason is the specificity of the sample tissue and pathogenic genes in  different  diseases. MMP2, as a member of the matrix metalloproteinase family, shows an increasing trend in the acute and chronic phases of lung disease. Our results observed the increased expression of MMP2 in asthma patients which is consistent with previous literatures [56].
Additionally, we identified the methylation status of the 9 aging-related genes in asthma patients. Most DMSs of asthma patients were hypermethylated, which was consistent with the differential expression of mRNA, indicating that DNA methylation regulating gene expression is related to aging. Moreover, except for ATG3, HDAC1, and TGFB1, correlation analysis showed that the expression of the aging-related genes in peripheral blood of asthma patients was associated with pulmonary function parameters (FEV1%, FEV1, FVC, PEF, FEF75, FEF50, FEF25). It is known that TGFB1 was a key cytokine that directs airway remodeling [57] and HDAC1 played a critical role in the pathogenesis of asthma [58]. This partial difference may be due to the presence of single nucleotide polymorphism in asthma [59]. Chr16:55514392 located in the promoter region has a regulatory effect on gene expression, which is inversely associated with lung function index (FVC) [60]. Interestingly, Chr16:55514437 is also located at the transcription initiation site, but the specific molecular mechanism which regulate gene expression still needs further study [60]. Furthermore, there were 9 asthma-related CpG sites on the CpG islands of the differential aging-related genes. The ROC curve and PCA analysis of methylation level showed that all the 9 DMSs could be used as potential biomarkers to distinguish asthma from HCs. Most notably, the methylation rate of either single DMS or total 9 DMSs in asthma patients were significantly higher than that of HCs. As population size and ethnicity may influence the methylation level, we assumed that a methylation marker
holds promise for better biomarker of asthma. Our analysis of the 9 DMSs methylation mutation rate also produced a better ROC specificity and sensitivity, suggesting that the combinatorial DMSs had a great potential to predict asthma. BALF (IL-25 and IL-33, etc.), induced sputum (eosinophils, Th2 cells, etc.) and airway remodeling could all be used as an useful indicators for asthma diagnosis [61, 62]. However, the detect of DNAm in peripheral blood has greater advantage of widespread access to samples and simple operation. Not only that, DNAm is an important cause of asthma exacerbation, the specific role of allergens and environmental exposure on the epigenetic modification during the exacerbation of asthma also deserved more attention [63].
Although our study provides potential value for diagnosis and treatment of asthma assessment, there are also some limitations. Firstly, asthma can be divided into different phenotypes which may have differential epigenetic modification. Besides, our previous work is not comprehensive enough to screen all the aging-related genes. Moreover, the sample size is relatively small.