Screening of key biomarkers and immune infiltration in Pulmonary Arterial Hypertension via integrated bioinformatics analysis

Figures & data

Figure 1. The workflow of this study

Table 1. Details of three GEO datasets

Dataset	Tissue	Platform	PAH	Normal	Reference (PMID)
GSE15197	lung	GPL6480	18	13	20,081,107
GSE113439	lung	GPL6244	15	11	30,963,672
GSE117261	lung	GPL6244	58	25	30,562,042

Note: GEO, Gene Expression Omnibus; PAH, Pulmonary arterial hypertension

Table 2. Screening DEGs in PAH by integrated analysis of microarray

DEGs	Gene names
Up-regulated	LTBP1, HBB, ACE2, SECISBP2L, PDE4D, ABCC9, PDE3A, TSHZ2, WIF1, DLG2, ITGB6, PDE7B, FREM1, EPHA4, MACC1, MALL, POSTN, IGF1, HIVEP2, N4BP2, ZFX, PLCB1, SFRP2, PI15, KLHL4, MACF1, PDE1A, PDE8B, ABCG2, ACADL, PREX2, CA1, PLCB4, IQGAP2, XAF1, ANKRD36B, FGFR2, INHBA, RGS5, TXLNG, ECM2, NT5E, ETV5, RASEF, LRRC36, VPS13A, FGD4, GEM, ANKRD36, MXRA5, CFH, ZNF521, CA2, C5, PAMR1, BMP6, GFRA1, RSPO3, THY1, PIEZO2, CCL21, DCLK1, ANKRD50, ALAS2, GBP5, SLC4A7, OGN, SULF1, NR1D2, SYNPO2, RGS1, ASPN, EML4, TFPI2, VCAM1, KIT, WEE1, ABCB1, HLTF, ANGPT2, RASGRP1, ITGB3, PSD3, CCL5, HMCN1, ITGA2, CCDC80, IL13 RA2, EPHA3, FABP4, HBD, CD5L, LRRC17, PHEX, GZMK, ENPP2, ESM1, PDGFD, TTN, MME, TFCP2L1, CD69, EYA4, NCKAP5, CXCL9, EDN1, SEMA3D, PKP2, IDO1, FAP, CPB2, ANKRD22, FMO5, SFRP4, PPBP, AREG, IGHA1
Down-regulated	RNASE2, CSF3R, GIMAP6, ADRA1A, LILRA2, GLT1D1, ITGAM, MGAM, NKD1, TBX3, S100A9, S100A8, LILRB2, SOSTDC1, CD14, SAA2, NQO1, QPCT, TLR8, SLC9A3R2, KRT4, CXCR1, AQP9, AGTR1, GALNT13, SLCO4A1, RNF182, VNN2, S100A12, S100A3, BPIFA1, SULT1B1, USP9Y, ZFY, IL1R2, SLCO2A1, LRRC32, SAA1, BTNL9, TXNRD1, MNDA, UTY, MS4A15, CR1, EIF1AY, CDH13, LRRN4, CXCR2, PROK2, KDM5D, VIPR1, BPIFB1, CHL1, CA4, SERPINA3, CHIT1, LCN2, MMP8, FAM107A, DDX3Y, OLFM4, FCN3, RPS4Y1, PLA2G7, HMOX1

DEGs, differentially expressed genes; PAH, Pulmonary arterial hypertension.

Figure 2. Identification of DEGs from three mRNA expression datasets. (a) Volcano plot of three mRNA expression datasets after integrated as one via R software. log FC, log2 Fold Change. (b) Heatmap of differentially expressed gene expression. The heatmap was generated using pheatmap package in R. The expression profiles greater than the mean are colored in red and those below the mean are colored in green. PAH, Pulmonary arterial hypertension

Table 3. GO analysis of DEGs in PAH

Category	Term	Count	P-value	FDR
BP	neutrophil chemotaxis	10	1.94E-08	2.62E-05
BP	inflammatory response	19	5.34E-08	3.61E-05
BP	positive regulation of smooth muscle cell proliferation	8	2.28E-06	1.03E-03
BP	cell chemotaxis	8	3.94E-06	1.33E-03
BP	positive regulation of inflammatory response	8	8.61E-06	2.33E-03
CC	extracellular space	47	1.54E-14	2.73E-12
CC	extracellular region	48	2.31E-12	2.04E-10
CC	cell surface	19	4.97E-06	2.93E-04
CC	extracellular exosome	48	6.73E-05	2.98E-03
CC	extracellular matrix	11	5.97E-04	2.11E-02
MF	integrin binding	9	1.04E-05	2.26E-03
MF	3ʹ,5ʹ-cyclic-AMP phosphodiesterase activity	5	1.21E-05	2.26E-03
MF	calcium ion binding	20	1.05E-04	1.31E-02
MF	heparin binding	8	1.15E-03	8.01E-02
MF	growth factor activity	8	1.24E-03	8.01E-02

Note: GO, Gene Ontology; DEGs, differentially expressed genes; PAH, Pulmonary arterial hypertension; BP, biological process; CC, cellular component; MF, molecule function; FDR, false discovery rate

Table 4. KEGG enrichment analysis of DEGs in PAH

Category	Term	Count	P-value	FDR
hsa04640	Hematopoietic cell lineage	9	1.33E-05	1.83E-03
hsa04060	African trypanosomiasis	5	7.71E-04	5.28E-02
hsa05418	Rap1 signaling pathway	9	5.22E-03	2.39E-01
hsa04061	Renin secretion	5	8.86E-03	2.60E-01
hsa05144	Chemokine signaling pathway	8	9.48E-03	2.60E-01
hsa04614	Cytokine-cytokine receptor interaction	9	1.22E-02	2.78E-01
hsa04657	Hypertrophic cardiomyopathy	5	1.74E-02	3.33E-01
hsa04062	Nitrogen metabolism	3	1.94E-02	3.33E-01
hsa04064	Dilated cardiomyopathy	5	2.22E-02	3.38E-01
hsa05143	Morphine addiction	5	2.88E-02	3.91E-01

Note: KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; PAH, Pulmonary arterial hypertension; FDR, false discovery rate

Figure 3. Top 10 enriched GO terms and top 10 KEGG pathways of differentially expressed genes. (A‑C) GO term enrichment analysis for (a) biological process, (b) molecular function, (c) cellular component. (d) KEGG pathway analysis. Node size represents gene ratio; node color represents P-value. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

Figure 4. Construction of the PPI network. (a) The nodes represent proteins, and the edges represent the interaction of proteins, while blue and red circles indicate downregulated and upregulated DEGs, respectively. (b) The only one module in the PPI network. The nodes represent proteins, and the edges represent the interaction of proteins, while blue and red circles indicate downregulated and upregulated DEGs, respectively

Figure 5. GO and KEGG analyses of module genes. (a) GO term enrichment analysis of module genes. (b) KEGG pathway analysis of module genes. Node size represents gene ratio; node color represents P_adj-value. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

Figure 6. A model for predicting PAH. (a) LASSO model. (b) ROC curves analysis of training set. (c) ROC curves analysis of test set. AUC, area under the curve. PAH, Pulmonary arterial hypertension

Figure 7. The bar plot visualizing the relative percent of 22 immune cell in each sample. Different colors represent different types of immune cells

Figure 8. The difference of immune infiltration between PAH samples and normal control samples. Blue, normal controls group; Red, PAH group. PAH, Pulmonary arterial hypertension