Identification of DEGs
A total of 1422 DEGs were identified in GSE73953, including 952 up-regulated genes and 470 down-regulated genes. The volcano plot of all DEGs and heat map of the top 50 up-regulated genes and the top 50 down-regulated genes are shown in Figures 1 and 2, respectively.
Pathway and GO enrichment analysis of DEGs
GO enrichment and KEGG pathway analysis was performed with DAVID, which consists of three terms, as shown in Figure 3(a), (b), (c), and (d).
The top 15 biological process (BP) of DEGs in GSE115857 was clustered in regulation of transcription from RNA polymerase II promoter (GO:0006357, 54DEGs), positive regulation of nuclear-transcribed mRNA poly(A) tail shortening (GO:0060213, 7DEGs), cell adhesion (GO:0007155, 48DEGs), positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay (GO:1900153, 6DEGs), response to cytokine (GO:0034097,11DEGs), extracellular matrix organization (GO:0030198,25DEGs), signal transduction (GO:0007165,99DEGs), cellular response to fibroblast growth factor stimulus (GO:0044344, 8DEGs), cell migration (GO:0016477,22DEGs), positive regulation of I-kappa B kinase/NF-kappa B signaling (GO:0043123,21DEGs), response to drug (GO:0042493,33DEGs), response to organic cyclic compound (GO:0014070,10DEGs), response to mechanical stimulus (GO:0009612,11DEGs), positive regulation of positive chemotaxis (GO:0050927,5DEGs), and negative regulation of cell proliferation (GO:0008285,40DEGs) (Table 1).
The top 15 cellular component (CC) of DEGs in GSE115857 was clustered in postsynaptic density (GO:0014069,27DEGs), cytosol (GO:0005829,246DEGs), nucleus (GO:0005634,380DEGs), nucleoplasm (GO:0005654,207DEGs), endoplasmic reticulum (GO:0005783,72DEGs), focal adhesion (GO:0005925,37DEGs), membrane (GO:0016020,160DEGs), CCR4-NOT complex (GO:0030014,5DEGs), SNARE complex (GO:0031201,9DEGs), cell projection (GO:0042995,11DEGs), nuclear membrane (GO:0031965,23DEGs), mitochondrion (GO:0005739,99DEGs), extracellular space (GO:0005615,100DEGs), phagocytic vesicle (GO:0045335,7DEGs), intracellular (GO:0005622,98DEGs), and mast cell granule (GO:0042629,5DEGs).
The top 15 molecular function (MF) of DEGs in GSE115857 was clustered in protein binding (GO:0005515,620DEGs), transcription factor activity, sequence-specific DNA binding (GO:0003700,92DEGs), identical protein binding (GO:0042802,68DEGs), neurotrophin TRKA receptor binding (GO:0005168,4DEGs), ATPase activity, coupled to transmembrane movement of substances (GO:0042626,9DEGs), steroid hormone receptor activity (GO:0003707,10DEGs), sequence-specific DNA binding (GO:0043565,47DEGs), transcription factor activity, RNA polymerase II core promoter proximal region sequence-specific binding (GO:0000982, 6DEGs), transcription factor binding (GO:0008134,29DEGs), zinc ion binding (GO:0008270,93DEGs), syntaxin-1 binding (GO:0017075,5DEGs), palmitoyl-CoA hydrolase activity (GO:0016290,4DEGs), anion transmembrane-transporting ATPase activity (GO:0043225,4DEGs), RNA polymerase II activating transcription factor binding (GO:0001102,7DEGs), and beta-catenin binding (GO:0008013,11DEGs).
The top 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of DEGs in GSE115857 was clustered in Rheumatoid arthritis (hsa05323,15DEGs), ABC transporters (hsa02010,10DEGs), TNF signaling pathway (hsa04668,16DEGs), SNARE interactions in vesicular transport (hsa04130,8DEGs), HTLV-I infection (hsa05166,28DEGs), Ubiquitin mediated proteolysis (hsa04120,17DEGs), Salmonella infection (hsa05132,12DEGs), Cell adhesion molecules (CAMs) (hsa04514,17DEGs), Osteoclast differentiation (hsa04380,16DEGs), NF-kappa B signaling pathway (hsa04064,12DEGs), PI3K-Akt signaling pathway (hsa04151,32DEGs), ECM-receptor interaction (hsa04512,11DEGs), AMPK signaling pathway (hsa04152,14DEGs), Amphetamine addiction (hsa05031,9DEGs), and Chagas disease (American trypanosomiasis) (hsa05142,12DEGs) (Table 2).
PPI network and hub gene analysis
To explore the relationship between these DEGs and to identify hub genes, a PPI network of DEGs was constructed using the STRING online database and visualized using Cytoscape. There were 1199 nodes and 5490 edges in the PPI network, including 760 up-regulated genes, 350 down-regulated genes. In addition, 6 modules from the PPI network were selected using the MCODE plug-in of Cytoscape: module 1 (score=24), consisting of 24 nodes and 276 edges (Figure 4 (a)), module 2 (score=13.04), consisting of 26 nodes and 163 edges (Figure 4 (b)), module 3 (score=8.078), consisting of 52 nodes and 206 edges (Figure 4 (c)), module 4 (score=5.971), consisting of 71 nodes and 209 edges (Figure 4 (d)), module 5 (score=5.051), consisting of 60 nodes and 149 edges (Figure 4 (e)), module 6 (score=4.654), consisting of 53 nodes and 121 edges (Figure 4(f)). Then, GO and pathway enrichment analysis of these module genes were performed by CluGo plug-in of Cytoscape.
For the BP, genes from module 1 were most significantly clustered in positive regulation of mitotic metaphase/anaphase transition (GO:0045842, 5DEGs), there is no BP enrichment in module 2, genes from module 3 were most significantly clustered in organelle fusion (GO:0048284, 15DEGs) and regulation of glomerular filtration (GO:0003093, 4DEGs), genes from module 4 were most significantly clustered in ribonucleoprotein complex export from nucleus (GO:0071426, 10DEGs), genes from module 5 were most significantly clustered in cell adhesion mediated by integrin (GO:0033627, 8DEGs) and maturation small subunit ribosomal RNA (SSU-rRNA) (GO:0030490, 6DEGs), genes from module 6 were most significantly clustered in pentose-phosphate shunt, oxidative branch (GO:0009051, 4DEGs) (Figure 5,Table 3).
For CC, genes from module 1 were most significantly clustered in cullin-RING ubiquitin ligase complex (GO:0031461, 12DEGs), genes from module 2 were most significantly clustered in clathrin-coated vesicle (GO:0030136, 13DEGs), genes from module 3 were most significantly clustered in SNARE complex (GO:0031201, 9DEGs), specific granule (GO:0042581, 15DEGs), phagocytic vesicle (GO:0045335, 11DEGs), and mast cell granule (GO:0042629, 4DEGs ) (Figure 6).
For MF, there is no MF enrichment in module 1, genes from module 2 were most significantly clustered in clathrin adaptor activity (GO:0035615, 3DEGs), genes from module 3 were most significantly clustered in SNAP receptor activity (GO:0005484, 7DEGs), glucocorticoid receptor binding (GO:0035259, 3DEGs ), and thrombin-activated receptor activity (GO:0015057, 3DEGs ), genes from module 4 were most significantly clustered in extracellular matrix structural constituent conferring tensile strength (GO:0030020, 5DEGs), genes from module 6 were most significantly clustered in phosphatidate phosphatase activity (GO:0008195, 3DEGs) (Figure 7).
For KEGG enrichment, genes from module 1 were clustered in ubiquitin mediated proteolysis (KEGG:04120, 15DEGs), genes from module 3 were clustered in soluble NSF Attachment Protein Receptor (SNARE) interactions in vesicular transport (KEGG:04130, 8DEGs), genes from module 4 were clustered in extracellular matrix (ECM)-receptor interaction (KEGG:04512, 8DEGs), there is no KEGG enrichment in module 2,5 and 6 (Figure 8, Table 4).
In the present study, we used cytoHubba to choose hub genes. According to the five classification methods in cytoHubba, the top 30 hub genes selected by these ranked methods in cytoHubba are shown in Table 5. Finally, three central genes were identified by overlapping the first 30 genes, as shown in Figure 9. VEGFA is the most excellent central genes based on five ranked methods. JUN and FOS were also selected as hub genes (Table 6).
GSEA identifies signaling pathways in IMN
We compared the data sets for IMN and living donors using GSEA to identify signaling pathways. The results indicated significant differences (FDR < 0.25, NOM P-value < 0.05) in the enrichment of the MSigDB collection (h.all.v7.1.symbols.gmt). We selected the most significantly enriched signaling pathways, based on normalized enrichment score (NES) (Figure 10, Table 7). The results indicated the data set with IMN was enriched for angiogenesis. 13 core genes were found, including PTK2, SLCO2A1, COL3A1, FSTL1, CCND2, SERPINA5, VEGFA, VAV2, OLR1, APP, POSTN, VCAN, and NRP1 (Figure 11).