Identification of DEMs in COPD Plasma
With an adjusted Pvalue < 0.05 and |logFC|>1.5 condition screening, 175 DEMs (96 upregulated and 79 downregulated) were identified in GSE31568 dataset, 34 DEMs (9 upregulated and 25 downregulated) were identified in GSE61741, and 1702 DEMs (623 upregulated and 1079 downregulated) were identified in GSE148153. The heatmaps of DEMs in the above three datasets are shown in Figure 1A-C, and the details of DEMs are listed in Supplementary Table S1-3. There were a total of 11 overlapping DEMs in three datasets (GSE31568, GSE61741 and GSE148153) analyzed by Venn diagram, among which 5 downregulated DEMs (miR-578, miR-142-3p, miR-375, miR-190b and miR-609) continued to change in the three datasets(Figure 1D and E).
Prediction of Upstream Transcription Factors of DEMs
In this study, FunRich software was used to predict the upstream transcription factors of candidate DEMs. The upstream transcription factors of downregulated DEMs are shown in Figure 2. For the downregulated DEM, the transcription factors were BARX1, SRF, HOXC9, SP1, LHX3, EGR1, POU2F1 and NKX6-1.
Prediction of Downstream Target Genes of DEMs
A total of 1074 genes targeted by candidate DEMs were predicted in the miRNet database. For better visualization, the DEM-target gene network in Figure 3 shows the corresponding relationship between the downregulated DEMs and its target genes. In addition, the number of target genes per DEM is listed in Table 1, and all predicted target genes are listed in Supplementary Table S4.
TABLE 1 Potential Target Genes of the Significantly Downregulated DEMs
Downregulated DEMs
|
Number
|
hsa-miR-578
hsa-miR-142-3p
hsa-miR-375
hsa-miR-190b
hsa-miR-609
|
150
389
477
62
52
|
Total
|
1074
|
GO and KEGG Analysis of Target Genes
Then, we performed GO and KEGG analysis on 1074 target genes of DEMs. As shown in Figure 4A and B, biological process (BP) analysis showed that DEM target genes were particularly rich in muscle tissue development, dephosphorylation and striated muscle tissue development; cellular component (CC) analysis showed that DEM target genes were mainly enriched in cell-substrate junction, cell-substrate adherens junction and focal adhesion; molecular function (MF) analysis showed that DEM target genes were significantly enriched in GTP binding, purine ribonucleoside binding, purine nucleoside binding, ribonucleoside binding and guanyl ribonucleotide binding. In addition, KEGG pathway analysis showed that DEM target genes were significantly enriched in PI3K-Akt signaling pathway, human papillomavirus infection, Salmonella infection, viral carcinogenesis and proteoglycans in cancer (Figures 5A and B).
Construction of PPI and DEM-Hub Gene Network
The PPI network of genes targeted by downregulated DEMs was established the STRING database. Then, we uploaded the above PPI network to Cytoscape and used its cytohubba plugin to screen for hub genes. Figure 6 showed the top 30 hub genes for downregulated DEMs. The top 10 hub genes were CTNNB1, TP53, STAT3, MAPK3, ESR1, MYC, PTEN, CASP3, IGF1R and HSP90AA1 (Table 2).
In order to better study the molecular mechanism of DEMs in AMI plasma, DEM- hub gene network was constructed by Cytoscape software. The interactions between downregulated DEMs and hub genes were as follows: miR-142-3p interacted with two hub genes, including CTNNB1, ESR1; miR-375 interacted with 8 hub genes, including CTNNB1, TP53, STAT3, MAPK3, MYC, CASP3, IGF1R, HSP90AA1; and miR-190b interacted with PTEN (Figure 7).
TABLE 2 Top 10 Hub Genes of the Significantly Downregulated DEMs in the PPI Network Ranked by MCC
Gene Symbol
|
Score
|
CTNNB1
TP53
STAT3
MAPK3
ESR1
MYC
PTEN
CASP3
IGF1R
HSP90AA1
|
80,510,912,850,188
80,510,613,293,604
80,507,020,030,922
80,500,373,410,997
80,496,134,514,956
80,495,506,368,530
80,334,559,846,470
80,308,302,995,002
79,464,776,547,042
75,533,943,191,505
|
Validation of Hub Genes Expression
According to DEM-hub gene network, the plasma levels of the top 10 hub genes (CTNNB1, TP53, STAT3, MAPK3, ESR1, MYC, PTEN, CASP3, IGF1R, and HSP90AA1) were then identified using the GSE66360 dataset. According to the significant hub gene screening criteria, for the screened downregulated DEM, compared with normal plasma, only the expression of STAT3 and PTEN increased continuously in AMI plasma, while the expressions of ESR1, MYC, and TP53 were the opposite (Figure 8A-J). Therefore, miR-375-STAT3 and miR-190b-PTEN were identified as two potential regulatory pathways in AMI plasma.