Data preprocessing
When analyzing GSE90074, we first judged the sample quality. After controlling for quality, all of the samples were well normalized (Supplementary Figure 1). After removing duplicate values and annotating probes, a total of 54560 expression probes obtained in our expression microarray yielded a matrix of 19537 gene expression values.
Weighted gene co-expression networks.
Before constructing the weighted co-expression network, we chose the soft-threshold β parameter as the suitable weighted parameter of the adjacency function. After calculation, we set soft-threshold β as 23 and selected a correlation coefficient close to 0.85 to construct gene modules (Figure 1A). In accordance with the basic idea of WGCNA, we determined the correlation matrix and adjacency matrix of the gene expression profile of all samples with the clinical features, and then converted them to a topological overlap matrix (TOM), which produced a system clustering tree of genes according to gene-gene non-ω similarity. Along with TOM, we performed hierarchical average linkage clustering to distinguish the gene modules of each gene network (deep split = 2, cut height = 0.25) (Figure 1C). In all samples, approximately eight gene modules were recognized in total by the dynamic tree cut (Figure 1B). The genes whose expression levels could not be correlated with clinical features were assigned to the gray module. Therefore, the genes within the gray module were not further analyzed. A heatmap of the topological overlap in the gene network is shown in Figure 1D.
Finding module of interest.
The heatmap showed numerous valued biological significance to identify modules with the most significant and associated clinical characteristics. As shown in Figure 2A, the most interesting associations in the module-feature relationship were the dark turquoise module and CAD (r2 = 0.22, P = 0.007) and the dark turquoise module and CAD_class (r2 = 0.2, P = 0.02). The dark turquoise module membership and gene significance were significantly negatively correlated (r2 = -0.52, P = 2.7E-09) (Figure 2D). Subsequently, we analyzed the relationship between the module and CAD class and found that the brown module and class 0 (r2 = -0.17, P = 0.04) and the yellow module and class 3 (r2 = -0.18, P = 0.03) had the most interesting associations (Figure 2B). The brown and yellow module membership and gene significance were significantly positively correlated (r2 = 0.25, P = 0.0014; r2 = 0.53, P = 8.6E-09, respectively) (Figure 2E and F). From the dark turquoise module, we identified 316 genes in total, and the brown and yellow modules had 244 genes in total. The details of the module genes are shown in Supplementary Tables 2 and 3, and a Venn diagram is shown in Figure 2C. The other module did not have enough relationships or statistical significance for further consideration.
Functional annotation.
To detect the biological relevance of module genes and function, all three module genes were subjected to GO functional, KEGG pathway and DO enrichment analyses. The results are shown in Figure 3. When analyzing GO functions, we identified 484 biological processes, 39 cellular components, and 22 molecular functions with an adjusted P < 0.05. Table 1 shows the top 10 items. Approximately 38 pathways were enriched in KEGG pathway analysis, and 147 DO items with an adjusted P < 0.05 (false discovery rate, FDR set at < 0.05) were identified. Table 2 shows the top 15 items.
Among these results, several items have been confirmed in previous studies that were associated with CAD, and these included neutrophil degranulation (GO:0043312); regulation of MAP kinase activity (GO:0043405); neutrophil activation involved in immune response (GO:0002283); neutrophil mediated immunity (GO:0002446); neutrophil activation (GO:0042119); atherosclerosis (DOID:2349); atherosclerosis (DOID:1936); arteriosclerotic cardiovascular disease (DOID:2348); cell adhesion molecules (CAMs) (hsa04514); cytokine-cytokine receptor interaction (hsa04060); fluid shear stress and atherosclerosis (hsa05418); rheumatoid arthritis (hsa05323); and NF-kappa B signaling pathway (hsa04064). The genes located in these items were selected for further analysis.
Table 1. GO analysis for genes (top 10 significantly enriched terms).
Item
|
ID
|
Description
|
P. adjust
|
geneID
|
BP
|
GO:0042119
|
neutrophil activation
|
2.94E-34
|
ALOX5/ARHGAP9/BIN2/BST1/CCL5/CD14/CD53/CFP/CXCR1/CXCR2/FCER1G/FCGR3B/FCN1/FGL2/FGR/FPR1/FPR2/HBB/HSPA6/HVCN1/IL18RAP/ITGAM/ITGAX/ITGB2/LILRA2/LILRA3/LILRB2/LILRB3/MCEMP1/MGAM/MMP25/MNDA/OSCAR/PECAM1/PPBP/PRAM1/PTAFR/PTPN6/RAB37/S100A12/S100A8/S100A9/SIGLEC5/SIRPB1/SLC11A1/STK10/TLR2/TNFRSF1B/TYROBP/ADAM8/ADGRE3/C5AR1/CD300A/CLEC4D/CTSS/FCGR2A/HSP90AB1/ITGAL/KMT2E/LRMP/MMP9/PGLYRP1/PLEKHO2/PRKCD/RAB24/SELL/SPTAN1/SYK/TBC1D10C
|
BP
|
GO:0002283
|
neutrophil activation involved in immune response
|
3.13E-32
|
ALOX5/ARHGAP9/BIN2/BST1/CD14/CD53/CFP/CXCR1/CXCR2/FCER1G/FCGR3B/FCN1/FGL2/FGR/FPR1/FPR2/HBB/HSPA6/HVCN1/ITGAM/ITGAX/ITGB2/LILRA2/LILRA3/LILRB2/LILRB3/MCEMP1/MGAM/MMP25/MNDA/OSCAR/PECAM1/PPBP/PRAM1/PTAFR/PTPN6/RAB37/S100A12/S100A8/S100A9/SIGLEC5/SIRPB1/SLC11A1/STK10/TLR2/TNFRSF1B/TYROBP/ADAM8/ADGRE3/C5AR1/CD300A/CLEC4D/CTSS/FCGR2A/HSP90AB1/ITGAL/LRMP/MMP9/PGLYRP1/PLEKHO2/PRKCD/RAB24/SELL/SPTAN1/SYK/TBC1D10C
|
BP
|
GO:0002446
|
neutrophil mediated immunity
|
8.39E-32
|
ALOX5/ARHGAP9/BIN2/BST1/CD14/CD53/CFP/CXCR1/CXCR2/FCER1G/FCGR3B/FCN1/FGL2/FGR/FPR1/FPR2/HBB/HSPA6/HVCN1/ITGAM/ITGAX/ITGB2/LILRA3/LILRB2/LILRB3/MCEMP1/MGAM/MMP25/MNDA/OSCAR/PECAM1/PPBP/PRAM1/PTAFR/PTPN6/RAB37/S100A12/S100A8/S100A9/SIGLEC5/SIRPB1/SLC11A1/STK10/TLR2/TNFRSF1B/TYROBP/ADAM8/ADGRE3/C5AR1/CD300A/CLEC4D/CTSS/FCGR2A/HSP90AB1/ITGAL/KMT2E/LRMP/MMP9/PGLYRP1/PLEKHO2/PRKCD/RAB24/SELL/SPTAN1/SYK/TBC1D10C
|
BP
|
GO:0043312
|
neutrophil degranulation
|
9.61E-32
|
ALOX5/ARHGAP9/BIN2/BST1/CD14/CD53/CFP/CXCR1/CXCR2/FCER1G/FCGR3B/FCN1/FGL2/FGR/FPR1/FPR2/HBB/HSPA6/HVCN1/ITGAM/ITGAX/ITGB2/LILRA3/LILRB2/LILRB3/MCEMP1/MGAM/MMP25/MNDA/OSCAR/PECAM1/PPBP/PRAM1/PTAFR/PTPN6/RAB37/S100A12/S100A8/S100A9/SIGLEC5/SIRPB1/SLC11A1/STK10/TLR2/TNFRSF1B/TYROBP/ADAM8/ADGRE3/C5AR1/CD300A/CLEC4D/CTSS/FCGR2A/HSP90AB1/ITGAL/LRMP/MMP9/PGLYRP1/PLEKHO2/PRKCD/RAB24/SELL/SPTAN1/SYK/TBC1D10C
|
BP
|
GO:0050900
|
leukocyte migration
|
9.49E-20
|
BST1/CCL4/CCL5/CCR7/CORO1A/CSF3R/CXCR1/CXCR2/DOK2/DYSF/FCER1G/FFAR2/FPR2/HCK/IL16/ITGAM/ITGAX/ITGB2/JAML/PECAM1/PF4/PPBP/PTAFR/PTPN6/S100A12/S100A8/S100A9/SELPLG/SIRPG/SLC7A7/STK10/TBX21/TNFSF14/TREM1/ADAM8/C5AR1/CD2/CD244/CD300A/CXCR3/DAPK2/GPSM3/IL17RA/IL1B/IL6R/ITGAL/LYN/MMP9/SELL/SYK/VAV1
|
BP
|
GO:0030595
|
leukocyte chemotaxis
|
7.16E-16
|
BST1/CCL4/CCL5/CCR7/CORO1A/CSF3R/CXCR1/CXCR2/DYSF/FCER1G/FFAR2/FPR2/IL16/ITGB2/JAML/PF4/PPBP/S100A12/S100A8/S100A9/TNFSF14/ADAM8/C5AR1/CXCR3/DAPK2/GPSM3/IL17RA/IL1B/IL6R/LYN/SYK/VAV1
|
BP
|
GO:0097530
|
granulocyte migration
|
1.31E-14
|
BST1/CCL4/CCL5/CCR7/CSF3R/CXCR1/CXCR2/DYSF/FCER1G/ITGB2/JAML/PECAM1/PF4/PPBP/S100A12/S100A8/S100A9/ADAM8/C5AR1/CD300A/DAPK2/IL17RA/IL1B/SYK/VAV1
|
BP
|
GO:0060326
|
cell chemotaxis
|
1.45E-14
|
BIN2/BST1/CCL4/CCL5/CCR7/CORO1A/CSF3R/CXCR1/CXCR2/DYSF/FCER1G/FFAR2/FPR2/IL16/ITGB2/JAML/PF4/PPBP/S100A12/S100A8/S100A9/TNFSF14/ADAM8/ARRB2/C5AR1/CXCR3/DAPK2/GPSM3/IL17RA/IL1B/IL6R/LYN/PRKCD/SYK/VAV1
|
BP
|
GO:1990266
|
neutrophil migration
|
2.12E-14
|
BST1/CCL4/CCL5/CCR7/CSF3R/CXCR1/CXCR2/DYSF/FCER1G/ITGB2/JAML/PECAM1/PF4/PPBP/S100A12/S100A8/S100A9/ADAM8/C5AR1/DAPK2/IL1B/SYK/VAV1
|
BP
|
GO:0006909
|
phagocytosis
|
1.90E-13
|
ARHGAP25/BIN2/CD14/CD300LF/CORO1A/DYSF/FCER1G/FCGR3A/FCN1/FGR/HCK/ITGAM/ITGB2/MARCO/NCF2/NCF4/PECAM1/SIRPB1/SIRPG/SLC11A1/TLR2/TMEM175/WAS/CD300A/CEACAM4/FCGR2A/HSP90AB1/IL1B/ITGAL/LYN/NCK1/PLCG2/PRKCD/SYK/VAV1/WIPF1
|
CC
|
GO:0030667
|
secretory granule membrane
|
5.17E-19
|
BST1/CD14/CD53/CXCR1/CXCR2/FCER1G/FCGR3B/FPR1/FPR2/HVCN1/ITGAM/ITGAX/ITGB2/LILRA3/LILRB2/LILRB3/MCEMP1/MGAM/MMP25/PECAM1/PTAFR/RAB37/SIGLEC5/SIRPB1/SLC11A1/STK10/TLR2/TNFRSF1B/TYROBP/ADAM8/ADGRE3/C5AR1/CD300A/CLEC4D/FCGR2A/ITGAL/LRMP/RAB24/SELL/TBC1D10C
|
CC
|
GO:0070820
|
tertiary granule
|
7.56E-16
|
CD53/CFP/FCER1G/FPR1/FPR2/HBB/ITGAM/ITGAX/ITGB2/LILRA3/LILRB2/MCEMP1/MGAM/OSCAR/PPBP/PTAFR/PTPN6/SIGLEC5/SLC11A1/ADAM8/ADGRE3/CD300A/CLEC4D/CTSS/MMP9/PGLYRP1/SPTAN1/TBC1D10C
|
CC
|
GO:0101002
|
ficolin-1-rich granule
|
1.07E-12
|
ALOX5/BIN2/FCER1G/FCN1/FGL2/FPR1/FPR2/HBB/HSPA6/ITGAX/ITGB2/LILRA3/LILRB2/MGAM/MNDA/SIGLEC5/SLC11A1/ADAM8/ADGRE3/CD300A/CLEC4D/CTSS/HSP90AB1/MMP9/PLEKHO2/TBC1D10C
|
CC
|
GO:0101003
|
ficolin-1-rich granule membrane
|
9.10E-11
|
FCER1G/FPR1/FPR2/ITGAX/ITGB2/LILRA3/LILRB2/MGAM/SIGLEC5/SLC11A1/ADAM8/ADGRE3/CD300A/CLEC4D/TBC1D10C
|
CC
|
GO:0070821
|
tertiary granule membrane
|
9.10E-11
|
CD53/FCER1G/FPR2/ITGAM/ITGAX/ITGB2/LILRA3/LILRB2/MCEMP1/MGAM/PTAFR/SIGLEC5/SLC11A1/ADAM8/CD300A/CLEC4D
|
CC
|
GO:0030139
|
endocytic vesicle
|
4.36E-09
|
CORO1A/DYSF/FCGR1B/GNLY/HBB/HLA-DRA/HLA-DRB4/HLA-DRB5/MARCO/NCF1/NCF2/NCF4/SLC11A1/TLR2/WAS/ADAM8/ARRB2/CD163/DVL2/HLA-DRB1/HLA-DRB3/LPAR2/PGLYRP1/RAB11FIP1/RAB24/SYK/TLR1
|
CC
|
GO:0042581
|
specific granule
|
5.40E-09
|
BST1/CD53/CFP/FPR2/HVCN1/ITGAM/ITGB2/LILRA3/MCEMP1/MMP25/OSCAR/PTPN6/RAB37/STK10/TNFRSF1B/ADAM8/CLEC4D/ITGAL/PGLYRP1/SPTAN1
|
CC
|
GO:0035579
|
specific granule membrane
|
2.13E-08
|
BST1/CD53/FPR2/HVCN1/ITGAM/ITGB2/LILRA3/MCEMP1/MMP25/RAB37/STK10/TNFRSF1B/ADAM8/CLEC4D/ITGAL
|
CC
|
GO:0060205
|
cytoplasmic vesicle lumen
|
1.28E-06
|
ALOX5/ARHGAP9/BIN2/CFP/DEFA3/F13A1/FCN1/FGR/GNLY/HBB/HSPA6/MNDA/OSCAR/PF4/PPBP/PTPN6/S100A12/S100A8/S100A9/SRGN/GHRL/HSP90AB1/PGLYRP1/PRKCD/SPTAN1
|
CC
|
GO:0031983
|
vesicle lumen
|
1.28E-06
|
ALOX5/ARHGAP9/BIN2/CFP/DEFA3/F13A1/FCN1/FGR/GNLY/HBB/HSPA6/MNDA/OSCAR/PF4/PPBP/PTPN6/S100A12/S100A8/S100A9/SRGN/GHRL/HSP90AB1/PGLYRP1/PRKCD/SPTAN1
|
MF
|
GO:0008329
|
signaling pattern recognition receptor activity
|
1.07E-06
|
CD14/FCN1/MARCO/PTAFR/TLR2/TLR8/PGLYRP1/TLR4
|
MF
|
GO:0038187
|
pattern recognition receptor activity
|
1.07E-06
|
CD14/FCN1/MARCO/PTAFR/TLR2/TLR8/PGLYRP1/TLR4
|
MF
|
GO:0004896
|
cytokine receptor activity
|
9.91E-05
|
CCR7/CSF3R/CXCR1/CXCR2/IL18RAP/IL1R2/IL2RB/CSF2RA/CXCR3/IL10RA/IL17RA/IL6R
|
MF
|
GO:0019864
|
IgG binding
|
0.00020022
|
FCER1G/FCGR1B/FCGR3A/FCGR3B/FCGR2A
|
MF
|
GO:0035325
|
Toll-like receptor binding
|
0.00020022
|
S100A8/S100A9/TLR2/SYK/TLR1
|
MF
|
GO:0019865
|
immunoglobulin binding
|
0.00061902
|
FCER1G/FCGR1B/FCGR3A/FCGR3B/LILRA2/FCGR2A
|
MF
|
GO:0019955
|
cytokine binding
|
0.00110874
|
CCR7/CSF1R/CSF3R/CXCR1/CXCR2/IL1R2/IL2RB/TNFRSF1B/CSF2RA/CXCR3/IL10RA/IL6R
|
MF
|
GO:0050786
|
RAGE receptor binding
|
0.00380145
|
FPR1/S100A12/S100A8/S100A9
|
MF
|
GO:0008528
|
G protein-coupled peptide receptor activity
|
0.00380145
|
CCR7/CXCR1/CXCR2/FPR1/FPR2/GALR3/KISS1R/OGFR/UTS2R/CXCR3/INPP5K/SIGMAR1
|
MF
|
GO:0001653
|
peptide receptor activity
|
0.00507011
|
CCR7/CXCR1/CXCR2/FPR1/FPR2/GALR3/KISS1R/OGFR/UTS2R/CXCR3/INPP5K/SIGMAR1
|
GO, gene ontology; BP, biological processes; CC, cellular components; MF, molecular functions
Table 2. KEGG and DO analysis for genes (top 15 significantly enriched terms).
Item
|
ID
|
Description
|
P. adjust
|
geneID
|
KEGG
|
hsa05150
|
Staphylococcus aureus infection
|
1.72E-10
|
DEFA3/FCGR3A/FCGR3B/FPR1/FPR2/HLA-DRA/HLA-DRB4/HLA-DRB5/ITGAM/ITGB2/PTAFR/SELPLG/C5AR1/FCGR2A/HLA-DRB1/HLA-DRB3/ITGAL
|
KEGG
|
hsa04380
|
Osteoclast differentiation
|
1.72E-10
|
CSF1R/FCGR3A/FCGR3B/LCP2/LILRA2/LILRA3/LILRA4/LILRB2/LILRB3/NCF1/NCF2/NCF4/OSCAR/SIRPB1/SIRPG/TYROBP/FCGR2A/IL1B/LILRA5/PLCG2/SPI1/SYK
|
KEGG
|
hsa05140
|
Leishmaniasis
|
3.34E-10
|
FCGR3A/FCGR3B/HLA-DRA/HLA-DRB4/HLA-DRB5/ITGAM/ITGB2/NCF1/NCF2/NCF4/PTPN6/TLR2/FCGR2A/HLA-DRB1/HLA-DRB3/IL1B/TLR4
|
KEGG
|
hsa05152
|
Tuberculosis
|
1.98E-09
|
BCL2/CD14/CLEC4E/CORO1A/FCER1G/FCGR3A/FCGR3B/HLA-DRA/HLA-DRB4/HLA-DRB5/ITGAM/ITGAX/ITGB2/TLR2/CTSS/FCGR2A/HLA-DRB1/HLA-DRB3/IL10RA/IL1B/LSP1/SYK/TLR1/TLR4
|
KEGG
|
hsa04060
|
Cytokine-cytokine receptor interaction
|
5.06E-07
|
BMP8B/CCL4/CCL5/CCR7/CSF1R/CSF3R/CXCR1/CXCR2/IL16/IL18RAP/IL1R2/IL2RB/LTB/OSM/PF4/PPBP/TNFRSF10C/TNFRSF1B/TNFSF14/CD27/CSF2RA/CXCR3/IL10RA/IL17RA/IL1B/IL6R/TNFSF10
|
KEGG
|
hsa04145
|
Phagosome
|
5.32E-07
|
CD14/CORO1A/FCGR3A/FCGR3B/HLA-DRA/HLA-DRB4/HLA-DRB5/ITGAM/ITGB2/MARCO/NCF1/NCF2/NCF4/TLR2/CTSS/FCGR2A/HLA-DRB1/HLA-DRB3/TLR4
|
KEGG
|
hsa04640
|
Hematopoietic cell lineage
|
8.96E-07
|
CD14/CD37/CSF1R/CSF3R/HLA-DRA/HLA-DRB4/HLA-DRB5/IL1R2/ITGAM/CD2/CSF2RA/HLA-DRB1/HLA-DRB3/IL1B/IL6R
|
KEGG
|
hsa04650
|
Natural killer cell mediated cytotoxicity
|
7.99E-06
|
FCER1G/FCGR3A/FCGR3B/GZMB/HCST/ITGB2/LCP2/PRF1/PTPN6/TYROBP/CD244/ITGAL/PLCG2/SYK/TNFSF10/VAV1
|
KEGG
|
hsa04062
|
Chemokine signaling pathway
|
5.75E-05
|
CCL4/CCL5/CCR7/CXCR1/CXCR2/FGR/HCK/NCF1/PF4/PPBP/RASGRP2/WAS/ARRB2/CXCR3/LYN/PLCB2/PRKCD/VAV1
|
KEGG
|
hsa05323
|
Rheumatoid arthritis
|
9.05E-05
|
CCL5/HLA-DRA/HLA-DRB4/HLA-DRB5/ITGB2/LTB/TLR2/HLA-DRB1/HLA-DRB3/IL1B/ITGAL/TLR4
|
KEGG
|
hsa05321
|
Inflammatory bowel disease (IBD)
|
0.00013055
|
HLA-DRA/HLA-DRB4/HLA-DRB5/IL18RAP/TBX21/TLR2/HLA-DRB1/HLA-DRB3/IL1B/TLR4
|
KEGG
|
hsa05332
|
Graft-versus-host disease
|
0.00016688
|
GZMB/HLA-DRA/HLA-DRB4/HLA-DRB5/PRF1/HLA-DRB1/HLA-DRB3/IL1B
|
KEGG
|
hsa04940
|
Type I diabetes mellitus
|
0.0002234
|
GZMB/HLA-DRA/HLA-DRB4/HLA-DRB5/PRF1/HLA-DRB1/HLA-DRB3/IL1B
|
KEGG
|
hsa04514
|
Cell adhesion molecules (CAMs)
|
0.00037834
|
CADM3/HLA-DRA/HLA-DRB4/HLA-DRB5/ITGAM/ITGB2/PECAM1/SELPLG/CD2/HLA-DRB1/HLA-DRB3/ITGAL/NTNG2/SELL
|
KEGG
|
hsa05330
|
Allograft rejection
|
0.00072889
|
GZMB/HLA-DRA/HLA-DRB4/HLA-DRB5/PRF1/HLA-DRB1/HLA-DRB3
|
DO
|
DOID:104
|
bacterial infectious disease
|
7.86E-12
|
ALOX5/BCL2/CCL4/CCL5/CCR7/CD14/CXCR2/DEFA3/FPR2/GZMA/HLA-DRB4/ITGAM/ITGAX/ITGB2/NLRP3/OSM/PECAM1/PRF1/PTAFR/SLC11A1/TLR2/TNFRSF1B/TREM1/C5AR1/CD27/FCGR2A/GHRL/HLA-DRB1/IL1B/ITGAL/MCL1/MMP9/SELL/TLR1/TLR4/TP53
|
DO
|
DOID:0050338
|
primary bacterial infectious disease
|
5.63E-10
|
ALOX5/BCL2/CCL4/CCL5/CCR7/CD14/CXCR2/DEFA3/GZMA/HLA-DRB4/ITGAX/NLRP3/PECAM1/PRF1/SLC11A1/TLR2/TNFRSF1B/TREM1/C5AR1/CD27/FCGR2A/GHRL/HLA-DRB1/IL1B/ITGAL/MCL1/MMP9/SELL/TLR1/TLR4/TP53
|
DO
|
DOID:399
|
tuberculosis
|
4.17E-09
|
ALOX5/BCL2/CCL4/CCL5/CD14/CXCR2/DEFA3/GZMA/ITGAX/NLRP3/PECAM1/PRF1/SLC11A1/TLR2/TNFRSF1B/CD27/HLA-DRB1/IL1B/ITGAL/MCL1/MMP9/TLR1/TLR4/TP53
|
DO
|
DOID:74
|
hematopoietic system disease
|
1.49E-07
|
BCAM/BCL2/CCL5/CSF3R/CXCR1/F13A1/FCGR3A/FCGR3B/HBB/HLA-DRA/ITGAM/ITGB2/PF4/PRF1/PTPN6/RASGRP4/SLC11A1/TBX21/TLR2/TLR8/TNFRSF1B/WAS/ZFPM1/CSF2RA/CXCR3/DCK/ETV6/FCGR2A/HLA-DRB1/HLA-DRB3/IL1B/MMP9/SELL/SPTAN1/TINF2/TLR4/TNFSF10/TP53/VAV1/WIPF1
|
DO
|
DOID:75
|
lymphatic system disease
|
5.22E-07
|
CCR7/FOXC2/GZMB/ITGAX/PECAM1/PRF1/S100A12/S100A8/S100A9/SLC11A1/CD163/HLA-DRB1/IL1B/ITGAL/MMP9/TLR4/TP53
|
DO
|
DOID:1936
|
atherosclerosis
|
5.22E-07
|
ALOX5/ALOX5AP/APOBR/CCL5/CD14/CXCR2/FCGR3A/FPR1/MNDA/NLRP3/PECAM1/PF4/S100A12/S100A8/S100A9/SOX18/TLR2/UTS2R/ADAM8/ADRB2/CD163/CTSS/CXCR3/FCGR2A/GHRL/IL1B/KLF2/MMP9/PGLYRP1/TLR4/TNFSF10/TP53
|
DO
|
DOID:2348
|
arteriosclerotic cardiovascular disease
|
5.22E-07
|
ALOX5/ALOX5AP/APOBR/CCL5/CD14/CXCR2/FCGR3A/FPR1/MNDA/NLRP3/PECAM1/PF4/S100A12/S100A8/S100A9/SOX18/TLR2/UTS2R/ADAM8/ADRB2/CD163/CTSS/CXCR3/FCGR2A/GHRL/IL1B/KLF2/MMP9/PGLYRP1/TLR4/TNFSF10/TP53
|
DO
|
DOID:2349
|
arteriosclerosis
|
9.86E-07
|
ALOX5/ALOX5AP/APOBR/CCL5/CD14/CXCR2/FCGR3A/FPR1/MNDA/NLRP3/PECAM1/PF4/S100A12/S100A8/S100A9/SOX18/TLR2/UTS2R/ADAM8/ADRB2/CD163/CTSS/CXCR3/FCGR2A/GHRL/IL1B/KLF2/MMP9/PGLYRP1/TLR4/TNFSF10/TP53
|
DO
|
DOID:850
|
lung disease
|
3.13E-06
|
BCL2/CCL5/CD14/CSTA/CXCR1/FCGR3B/FGL2/GZMA/HBB/HCK/IL16/IL18RAP/ITGAM/ITGB2/NCF1/NCF2/S100A9/SELPLG/SLC11A1/TBX21/TLR2/TREM1/ADAM8/ADRB2/C5AR1/CSF2RA/CTSS/CXCR3/FCGR2A/HLA-DRB1/IL10RA/IL1B/ITGAL/MMP9/NLRP1/PRKCD/TLR4/TP53
|
DO
|
DOID:3083
|
chronic obstructive pulmonary disease
|
6.69E-06
|
BCL2/CCL5/CD14/CSTA/FGL2/GZMA/HBB/HCK/IL16/ITGAM/ITGB2/SLC11A1/TLR2/TREM1/ADRB2/C5AR1/CTSS/CXCR3/IL10RA/IL1B/ITGAL/MMP9/TLR4
|
DO
|
DOID:3388
|
periodontal disease
|
6.69E-06
|
CCL5/CD14/CXCR2/DEFA3/FCGR3A/FCGR3B/FPR1/IL16/IL1R2/OSM/PECAM1/S100A8/TLR2/FCGR2A/IL1B/MMP9/TLR4/TNFSF10
|
DO
|
DOID:1247
|
blood coagulation disease
|
1.55E-05
|
CCL5/F13A1/FCGR3B/HLA-DRA/ITGAM/ITGB2/PF4/PRF1/TLR2/WAS/ZFPM1/FCGR2A/HLA-DRB1/HLA-DRB3/IL1B/MMP9/SELL/TLR4/TNFSF10/WIPF1
|
DO
|
DOID:2320
|
obstructive lung disease
|
1.56E-05
|
BCL2/CCL5/CD14/CSTA/FGL2/GZMA/HBB/HCK/IL16/IL18RAP/ITGAM/ITGB2/SELPLG/SLC11A1/TBX21/TLR2/TREM1/ADAM8/ADRB2/C5AR1/CTSS/CXCR3/IL10RA/IL1B/ITGAL/MMP9/TLR4
|
DO
|
DOID:824
|
periodontitis
|
1.81E-05
|
CCL5/CD14/CXCR2/DEFA3/FCGR3A/FCGR3B/FPR1/IL16/IL1R2/OSM/S100A8/TLR2/FCGR2A/IL1B/MMP9/TLR4
|
DO
|
DOID:1555
|
urticaria
|
1.81E-05
|
ALOX5/ALOX5AP/FPR2/NLRP3/PF4/PPBP/ADRB2/HLA-DRB1/MMP9/SYK
|
PPI network construction and identification of hub genes.
To elucidate the PPI network of these selected genes, we used the STRING database to perform the analysis. When the cutoff was set as a combined score > 0.9, approximately 3218 protein pairs and 285 nodes were included. Figure 4A shows the net analysis from Cytoscape. Four modules with a score > 10 were found and are presented in Figure 4 (B-E) for detection using the Molecular Complex Detection (MCODE) app.
These four modules included 49 genes in total. Finally, approximately 6 genes demonstrated a high degree of association simultaneously in the submodule analysis and were screened with GO, DO, and KEGG data. These six genes were toll-like receptor 2 (TLR2), CD14 molecule (CD14), NLR family pyrin domain containing 3 (NLRP3), C-X-C motif chemokine receptor 2 (CXCR2), C-C motif chemokine ligand 5 (CCL5) and platelet factor 4 (PF4).
We employed the GoSemSim package in R to analyze the interactions between the proteins and scored them according to their average functional similarity. Proteins with higher scores had higher functional interactions and warranted further investigation. TLR2 and CD14 were the two top-ranked proteins potentially playing central roles in the interaction (Figure 5).
At the same time, we found that the expression of TLR2 was higher in different classes of coronary artery lesions than in class 0, while the expression of CD14 was higher only in class 4 than in class 0. The expression of the other genes was not significantly different in all groups (Figure 6A). This result suggests that TLR2 and CD14 may be related to the severity of coronary atherosclerosis.
Hub gene validation.
The training data set (GSE60993) was used to verify the correlation between two hub genes (TLR2 and CD14) and atherosclerotic plaque vulnerability. We compared the expression of each candidate hub gene during the development of CAD (including stable CAD, unstable angina, NSTEMI and STEMI). One‐way analysis of variance test results showed that the expression of TLR2 was closely related to NSTEMI and STEMI, whereas the expression of CD14 was only closely related to NSTEMI. Patients with acute myocardial infarction had a high level of gene expression (Figure 6B).
Subsequently, we compared the gene expression levels of these two genes at different times (1 day, 4-6 days, 30 days and 180 days) after myocardial infarction. We found that in the TLR2 group, the expression of TLR2 increased significantly in AMI (1 day and 4-6 days after MI) but decreased in the convalescent period (30 days and 180 days after MI). Similar results were found in the CD14 group, but the expression level of CD14 in convalescence was still higher than that in the stable coronary heart disease group (Figure 6C).
To further explore these two genes and the occurrence of heart failure after myocardial infarction, we performed a two-factor survival analysis on GSE59867. According to the expression of TLR2 and CD14, the patients were divided into four groups according to the median value of TLR2 and CD14. Survival analysis of heart failure events after myocardial infarction was calculated. As shown in Figure 6D, we found that the incidence of heart failure events was the lowest when the expression levels of these two genes were both decreased, which was statistically significant compared with the other three groups (P < 0.01 for all).
As shown in Figure 7, we validated these data in a population sample and came to the same conclusion as the previous microarray data. Both expression levels of TLR2 and CD40 were lowest in the normal group and were high in the population with coronary artery disease and acute coronary syndrome.
Table 3. Comparison of demographic, lifestyle characteristics and serum lipid levels among different groups.
Parameter
|
Control
|
CAD
|
UA
|
NSTEMI
|
STEMI
|
Number
|
212
|
220
|
204
|
191
|
202
|
Male/female
|
44/174
|
39/181
|
44/160
|
34/157
|
48/154
|
Age (years)1
|
55.27±8.24
|
54.29±8.43
|
55.57±7.92
|
56.11±8.91
|
56.02±7.78
|
Height (cm)
|
166.13±6.74
|
167.63±7.10
|
167.49±7.26
|
166.52±6.88
|
167.09±8.74
|
Weight (kg)
|
52.46±6.88
|
54.72±9.93
|
58.61±8.21a
|
57.89±9.13a
|
58.12±9.32a
|
Body mass index (kg/m2)
|
29.21±5.23
|
30.24±6.57a
|
30.87±7.32a
|
30.89±7.14a
|
30.96±6.19a
|
Waist circumference (cm)
|
74.29±6.71
|
73.54±9.28
|
74.63±7.65
|
75.28±7.22
|
76.25±6.31
|
Smoking status [n (%)]
|
56(26.0)
|
79(35.7) a
|
93(45.8) a
|
92(48.4) a
|
99(48.9) a
|
Alcohol consumption [n (%)]
|
51(24.2)
|
57(25.7)
|
48(23.3)
|
56(29.2) a
|
58(28.8) a
|
SBP (mmHg)
|
123.15±16.22
|
129.32±22.16a
|
148.01±23.77c
|
141.45±23.14b
|
106.45±10.16c
|
DBP (mmHg)
|
81.54±11.23
|
81.34±12.01
|
89.04±15.21a
|
83.23±8.20
|
62.51±18.74c
|
PP (mmHg)
|
49.64±14.13
|
51.42±13.59
|
51.66±15.24
|
50.84±15.22
|
50.22±14.21
|
Glucose (mmol/L)
|
5.93±1.81
|
6.22±2.23
|
7.74±2.21b
|
8.55±2.31c
|
8.44±2.69c
|
TC (mmol/L)
|
4.95±1.32
|
5.26±1.28a
|
5.69±1.02a
|
5.99±1.24a
|
5.84±1.46a
|
TG (mmol/L)2
|
1.48(0.51)
|
1.52(1.21)
|
1.53(1.22)
|
1.46(1.41)
|
1.46(1.38)
|
HDL-C (mmol/L)
|
1.51±0.43
|
1.29±0.34a
|
1.31±0.31a
|
1.40±0.31a
|
1.43±0.35
|
LDL-C (mmol/L)
|
2.87±0.83
|
3.24±0.75a
|
3.99±0.81a
|
3.89±0.98a
|
3.96±0.81a
|
ApoA1 (g/L)
|
1.23±0.24
|
1.16±0.22
|
1.18±0.26
|
1.14±0.26
|
1.15±0.28
|
ApoB (g/L)
|
0.84±0.19
|
0.82±0.32
|
0.81±0.31
|
0.94±0.31
|
0.88±0.28
|
ApoA1/ApoB
|
1.68±0.49
|
1.67±0.56
|
1.65±0.52
|
1.66±0.63
|
1.68±0.55
|
Heart rate (beats/minutes)
|
72.43±10.22
|
72.36±10.34
|
77.21±10.54a
|
76.45±9.32a
|
79.78±10.25a
|
Creatinine, (μmol/L)
|
72.55±12.21
|
71.38±11.44
|
74.43±11.42
|
77.55±10.21
|
76.68±12.84
|
Uric acid, (μmol/L)
|
280.96±74.23
|
283.89±72.32
|
278.88±84.32
|
286.91±82.32
|
284.76±76.38
|
Troponin T, (μg/L)
|
0.01±0.02
|
0.02±0.02
|
0.02±0.01
|
2.74±3.93c
|
2.86±6.28c
|
CK, (U/L)
|
87.82±43.21
|
88.84±47.31
|
92.81±51.32
|
1211.93±688.32c
|
1224.89±793.18c
|
CKMB, (U/L)
|
11.41±3.53
|
12.11±2.88
|
11.22±3.42
|
131.87±63.45c
|
138.74±57.16c
|
CAD, coronary artery disease; UA, unstable angina; NSTEMI, on-ST-segment elevation myocardial infarction; STEMI, ST-segment elevation myocardial infarction; SBP, systolic blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Apo, Apolipoprotein. 1Mean ± SD determined by t-test.2Because of not normally distributed, the value of triglyceride was presented as median (interquartile range), the difference between the two groups was determined by the Wilcoxon-Mann-Whitney test. The P value was defined as the comparison of case and control groups. aP < 0.05; bP < 0.01; cP < 0.001.