Data preprocessing and screening for DEGs
Following data preprocessing, 4 subsets of DEGs were analyzed, namely; COPD vs. No-COPD, male-COPD vs. male-No-COPD, female-COPD vs. female-No-COPD and male-COPD vs. female -COPD). Thereafter, the study analyzed the global transcriptomic changes in the lung tissues of COPD patients. As a result, a total of 1,442 DEGs, including 808 up-regulated and 634 down-regulated genes, were identified in lung tissues of COPD patients, compared to the healthy controls (Figure 2A-B). The DEGs are listed in Supplementary Table S1. Furthermore, 658 up-regulated and 908 down-regulated genes were identified in the lung tissues of male COPD patients, compared to the healthy male controls. On the other hand, 576 up-regulated and 480 down-regulated genes were identified in the lung tissues of female COPD patients, compared to the healthy female controls. Additionally, 1,157 DEGs were identified in the lung tissues of male COPD patients compared to the female ones, including 489 up-regulated and 668 down-regulated genes. The heatmaps and volcano plot showing the gene expression profiles of DEGs are presented in Figure 3C-H. The results presented above therefore show that a considerable number of DEGs in both males and females, is regulated in the lung in response to COPD. The detailed information on DEGs is listed in Supplementary Table S2–4.
The study further investigated whether the genes that were either up-regulated or down-regulated between COPD patients and healthy controls were the same in males and females. Interestingly, only 1.1% (13 out of 1221) of the genes that were up-regulated and 0.8% (11 out of 1377) of those that were down-regulated in COPD were shared between males and females (Figure 2I-J). The results therefore suggusted that most of the DEGs in male or female COPD patients were different, implicating a diffirent mechanism based on sex, in the disease. The overlapping DEGs are listed in Supplementary Table S5.
Functional annotation for DEGs between all the COPD patients and healthy control samples
The GO and KEGG analysis of DEGs were performed on Metascape. The results of Go enrichment analysis demonstrated that DEGs between the COPD samples and healthy controls were mainly enriched in “leukocyte migration”, “leukocyte activation involved in immune response”, “regulation of MAPK cascade”, “regulation of T cell activation” and “positive regulation of programmed cell death” (Figure 3A). Addtionally, several pathways, such as the “foxo signaling pathway”, “apoptosis”, “Retrograde endocannabinoid signaling”, “Cytokine-cytokine receptor interaction”, the “Chemokine signaling pathway”, “NOD-like receptor signaling pathway” and “p53 signaling pathway”, were enriched in the DEGs of COPD patients (Figure 3B). The results are demonstrated in Supplementary Table S6-7.
Functional annotation of the different subsets of DEGs in male and female COPD patients
The results revealed a clear difference in the GO and KEGG pathway terms between the male and female COPD patients (Figure 4). Notably, the DEGs in male patients were enriched in the GO terms related to “positive regulation of apoptotic process”, “negative regulation of phosphorylation” and “positive regulation of NK T cell activation” . On the other hand, the DEGs in female COPD patients were mainly enriched in “negative regulation of immune effector process” and “regulation of immune effector process” (Figure 4A-B). Interestingly, the DEGs in male COPD patients were enriched in such pathways as the “RIG-I-like receptor signaling pathway”, “GnRH signaling pathway”, “p53 signaling pathway” and “PI3K-Akt signaling pathway”. However, the DEGs in female COPD patients were enriched in pathways including “cholesterol metabolism”, “Glutamatergic synapse” and “Hippo signaling pathway” (Figure 4D-E). Additionally, the DEGs in both male and female COPD sampls were enriched in several GO terms, such as the “cellular lipid catabolic process”, “lipid biosynthetic process”, “hormone biosynthetic process” and “regulation of hormone levels” (Figure 4C). Moreover, the DEGs in both male and female COPD samples were mainly enriched in pathways such as the “PPAR signaling pathway”, “AMPK signaling pathway”, “Calcium signaling pathway” and “p53 signaling pathway” (Figure 4F). The results are demonstrated in Supplementary Table S8-13.
Construction of a PPI network for the enriched DEGs from KEGG analysis and selection of hub genes
We detected 14,211 genes associated with COPD from the Phenolyzer, and 121 genes of those genes belonged to seed genes(Supplementary Table S14). 336, 137 and 193 overlapping DEGs for male COPD patients compared to healthy male controls, female COPD patients compared to healthy female controls and male COPD patients compared to female COPD patients, were obtained between these 14,211 genes associated with COPD from the Phenolyzer and the enriched DEGs from KEGG analysis(Supplementary Table S15).
A PPI network of the different subsets of above overlapping DEGs was constructed in order to distinguish hub genes from the common one(Figure 5-7). The top ten degree genes in the PPI network were identified as the hub genes. The following were identified as the hub genes in the lung tissues of male COPD patients compared to healthy male controls; NRAS, RAC1, ATG7, UBE2N, SNRPF, DHX38, SNRPD1, HNRNPC, SRSF5 and CPSF1. On the other hand, the following were identified as the hub genes in the lung tissues of female COPD patients compared to healthy female controls; APOE, GNGT2, FGA, FGG, ADCY2, C5, HEXB, PDYN, APOC3 and PLA2G7. Notably, the analysis revealed that male and female COPD patients had different key genes. In addition, UBE2N, CDK2, ANAPC2, CDC16, ANAPC4, ANAPC7, UBE2R2, HUWE1, UBE4A and KLHL13 were identified as the unique hub genes between the male and female COPD patients.
Construction of the hub genes-pathway network
The hub genes-pathway network for the different subsets was constructed to further explore the key signaling pathways in which the selected hub genes were involved (Figure 8). Therefore, using the degree method in cytoHubba, the 10 genes with the highest degrees were identified as hub genes in the network. In the PPI network, it was shown that one hub gene was associated with different pathways while one pathway connected to several hub genes. In addition, the results showed that NRAS and RAC1 functioned through the “Rap1 signaling pathway” and “PI3K-Akt signaling pathway”, in male COPD patients. This suggested that NRAS, RAC1, the “Rap1 signaling pathway” and “PI3K-Akt signaling pathway” played an important in the mechanisms of COPD in male patients (Figure 8A and Supplementary Figure 1). On the other hand, “Cholesterol metabolism” was one of the important pathways in female COPD patients and the hub genes, APOE and APOC3, functioned through “Cholesterol metabolism” (Figure 8B and Supplementary Figure 2). Moreover, “Ubiquitin mediated proteolysis” and the “p53 signaling pathway” played more important roles in male COPD patients compared to the females. Furthermore, CDK2 and UBE2N were the hub genes involved in the “p53 signaling pathway” and “Ubiquitin mediated proteolysis”, respectively (Figure 8C and Supplementary Figure 3).
In addition, the CTD database showed that NRAS, RAC1, APOE, APOC3, UBE2N and CDK2 involved several respiratory tract diseases including COPD (NRAS: inference score 26.9; RAC1: inference score 36.5; APOE: inference score 30.57; APOC3: inference score 13.71; UBE2N: inference score 19.94; CDK2: inference score 15.99). This data is shown in Figure 9.