Novel hub genes associated with pulmonary artery remodeling in pulmonary hypertension

Abstract

Pulmonary hypertension (PH) is a life-threatening disease with complex pathogenesis. According to etiology, PH is divided into five major groups in clinical classification. However, pulmonary artery (PA) remodeling is their common feature, in addition to bone morphogenetic protein receptor type 2; it is elusive whether there are other novel common genes and similar underlying mechanisms. To identify novel common hub genes involved in PA remodeling at different PH groups, we analyzed mRNA-Seq data located in the general gene expression profile GSE130391 utilizing bioinformatics technology. This database contains PA samples from different PH groups of hospitalized patients with chronic thromboembolic pulmonary hypertension (CTEPH), idiopathic pulmonary artery hypertension (IPAH), and PA samples from organ donors without known pulmonary vascular diseases as control. We screened 22 hub genes that affect PA remodeling, most of which have not been reported in PH. We verified the top 10 common hub genes in hypoxia with Sugen-induced PAH rat models by qRT-PCR. The three upregulated candidate genes are WASF1, ARHGEF1 and RB1 and the seven downregulated candidate genes are IL1R1, RHOB, DAPK1, TNFAIP6, PKN1, PLOD2, and MYOF. WASF1, ARHGEF1, and RB1 were upregulated significantly in hypoxia with Sugen-induced PAH, while IL1R1, DAPK1, and TNFA1P6 were upregulated significantly in hypoxia with Sugen-induced PAH. The DEGs detected by mRNA-Seq in hospitalized patients with PH are different from those in animal models. This study will provide some novel target genes to further study PH mechanisms and treatment.

Keywords: bioinformatic analysis; chronic thromboembolic pulmonary hypertension; idiopathic pulmonary artery hypertension; pulmonary artery remodeling; pulmonary hypertension.

Figure 1

Identification and comparative analysis of DEGs in IPAH and CTEPH. (A,B) Volcano plot analysis identifying DEGs. Red denotes upregulated gene and blue signifies downregulated gene from PA samples from patients with IPAH and CTEPH (|logFC| > 1, adj. p < 0.05). (C) Downregulated and upregulated DEGs overlap between IPAH and CTEPH by the bioinformatic analysis were illustrated in the Venn diagram. (D) The detail of three special overlapping DEGs between IPAH and CTEPH.

Figure 2

Biological features in IPAH and CTEPH. (A,B) A part of the GO enrichment results shows the DEGs in IPAH (adj. p < 0.05 and q < 0.2). (C,D) A part of the GO enrichment results showing the DEGs in CTEPH (adj. p < 0.05 and q < 0.2). (E,F) The overlapping GO of DEGs between IPAH and CTEPH in the Venn diagram and the detail of 22 overlapping GOs of DEGs between IPAH and CTEPH. BP, biological processes; CC, cellular components, and MF, molecular functions.

Figure 3

KEGG pathways in IPAH and CTEPH. (A,B) KEGG pathways analysis of DEGs in IPAH (adj. p < 0.05 and q < 0.2). (C) KEGG pathways analysis of DEGs in CTEPH (gene count >2 and p < 0.05). (D) The overlapping KEGG pathways of DEGs between IPAH and CTEPH in the Venn diagram and the detail of five overlapping KEGG pathways of DEGs between IPAH and CTEPH.

Figure 4

PPI networks and modules in IPAH and CTEPH. (A) STRING software was used to analyze the PPI network of 1,305 DEGs in IPAH (left). There were 1,080 nodes and 4,940 edges. Cytohubba identified the top 10 hub genes in IPAH (right). (B) STRING software was used to analyze the PPI network of 306 DEGs in CTEPH (left). There were 274 nodes and 285 edges. Cytohubba identified the top 10 hub genes in CTEPH (right).

Figure 5

Common DEGs in IPAH and CTEPH and the hub genes of common DEGs. (A) Heatmap of the common 120 DEGs in IPAH and CTEPH. Red areas denote increased gene expressions, and blue areas denote decreased gene expressions in PA compared to normal. (B) In the correlation heatmap, abscissa and ordinate indicate 22 DEGs features by Spearman correlation analysis. The absolute value of the correlation coefficient is displayed on the right side of the color scale (from −1 to 1, and from blue to red). (C,D) GO enrichment result of the common DEGs. (E,F) KEGG pathway analysis of the common DEGs. (G) The most significant modules are identified in IPAH and CTEPH by Cytoscape.

Figure 6

The expression analysis of 22 common hub DEGs in human lung and smooth muscle tissues. (A) Gene expression level of hub genes in HPA database. (B) Gene expression scores of hub genes were compared in the Bgee database. (C) Protein expression of hub genes in HPA database.

Figure 7

Verification of the top 10 hub genes in hypoxia with Sugen-induced PAH rats by qRT-PCR. (A–D) Hypoxia with Sugen-induced PAH (SuHx) characterized by increased mean pulmonary artery pressure [mPAP, (A)], pulmonary vascular resistance [PVR, (B)], hypertrophy of right ventricle [RV/(LV+S), (C)] and pulmonary artery [wall thickness, (D)]. Data are the mean ± SE, and t-test (and non-parametric tests) was used for two Comparisons. n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001, as compared with control (control rats). (E) The gene expression of the top 10 hub genes in the pulmonary artery was verified by qRT-PCR. N = 3, * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 8

The protein expression of RB1, ARHGEF1, and WASF1 in model rat PA tissues. (A) Representative Western blot bands of RB1, ARHGEF1, and WASF1 in control and SuHx. (B) Integrated density analysis of the protein levels of RB1, ARHGEF1, and WASF1 in each group. n = 3, * p < 0.05.