Integrative analyses of gene expression profile reveal potential crucial roles of mitotic cell cycle and microtubule cytoskeleton in pulmonary artery hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is a life-threatening condition. The aim of this study was to explore potential crucial genes and pathways associated with PAH based on integrative analyses of gene expression and to shed light on the identification of biomarker for PAH.

Methods: Gene expression profile of pulmonary tissues from 27 PAH patients and 22 normal controls were downloaded from public database (GSE53408 and GSE113439). After the identification of differentially expressed genes (DEGs), hub pathways and genes were identified based on the comprehensive evaluation of protein-protein interaction (PPI) network analysis, modular analysis and cytohubba's analysis, and further validated in another PAH transcriptomic dataset (GSE33463). Potentially associated micro-RNAs (miRNAs) were also predicted.

Results: A total of 521 DEGs were found between PAH and normal controls, including 432 up-regulated DEGs and 89 down-regulated DEGs. Functional enrichment analysis showed that these DEGs were mainly enriched in mitotic cell cycle process, mitotic cell cycle and microtubule cytoskeleton organization. Moreover, five key genes (CDK1, SMC2, SMC4, KIF23, and CENPE) were identified and then further validated in another transcriptomic dataset associated with special phenotypes of PAH. Furthermore, these hub genes were mainly enriched in promoting mitotic cell cycle process, which may be closely associated with the pathogenesis of PAH. We also found that the predicted miRNAs targeting these hub genes were found to be enriched in TGF-β and Hippo signaling pathway.

Conclusion: These findings are expected to gain a further insight into the development of PAH and provide a promising index for the detection of PAH.

Keywords: Differentially expressed gene; Functional enrichment analysis; Protein-protein interaction network; Pulmonary arterial hypertension; miRNAs.

Fig. 1

Differential expression analyses for all genes between PAH and control. a. Principal component analysis (PCA) illustrated the individual differences in the Chip expression profiles among the PAH and control groups in 49 samples. The green and red circles represent the case and control groups, respectively. The first component represent 42.84% of variance and the second component represent 9.57% of variance. The PCA plot was plotted using prcomp function in R program. b. Volcano plot showed all the gene expression change in PAH compared to the control samples. Grey represents no change in expression, blue represents downregulation (Down), and red represents upregulation (Up). Log FC reprsents log2 fold changes and p-value < 0.05 was considered as the threshold value of significant difference. The cutoff of log FC was 1.0. c. Heatmap showed the top 100 differentially expressed genes listed by listed by corrected P-values in PAH compared to the control samples. Each column represents one sample, and each row represents one gene. The gene expression values of all samples are showed as base − 2 logarithmic value. The gradual color ranging from blue to red represents the changing process from downregulated to upregulated expression

Fig. 2

GO term enrichment analysis for DEGs revealed several biological processes associated with PAH. ClueGo network analysis was performed for all differentially expressed genes(a), up-regulated DEGs (b) and down-regulated DEGs (c). Enrichment for GO groups was conducted using the ClueGO plug-in. Nodes were colored according to grouping of related functions by statistically significant association of related GO terms. In each group, only the most significant term was labeled, and the node size corresponded to the significance of each GO term

Fig. 3

Identification of hub genes from DEGs. a. The protein–protein interaction (PPI) network of top 100 DEGs. Red nodes represent up-regulated DEGs and green nodes represent down-regulated DEGs. The size of circles represents levers of degree (number of interaction) and the thickness of edge represent strength of interaction with combined score. b. PPI network of the genes enriched in Mitotic Cell Cycle and Microtubule Cytoskeleton Organization. The nodes represented genes, blue indicated the DEGs shared by the two biological processes and the other colors corresponded to the genes in these two biological processes. And the size of nodes indicated the number of connections. Edges denoted the interactions between two genes, and the width of an edge denoted the score of a physical interaction. c. The core module (module 1 with the MCODE score of 29.067) from the PPI network. The color shadow of each node represents the Mcode_score (degree of connection of nodes). d. Venn diagram indicated 9 hub genes overlapping from three analytic procedure (PPI, Modular and Cytohub)

Fig. 4

Violin diagram showing the expression levels of five hub genes are highly corrected with PAH, especially with IPAH and SSc-PAH. These hub genes include CHEK1 (a-b), SMC2 (c-d), SMC4 (e-f), KIF23 (g-h) and CENPE (i-j). P-values were respectively obtained from two-sample Wilcoxon test and multiple-sample Kruskal−Wallis test

Fig. 5

Functional association of five hub genes. a. Clustering heatmap of 5 hub genes. “Red” indicates high relative expression, and “Blue” indicates low relative expression.b. Correlations analysis of hub genes. “Red” represents positive correlation and “Blue” represents negative correlation. The size of circle and number indicates correlation coefficient. c. Pie Chart indicates distribution of respiratory systemic disease correlative with hub genes. Each module with color represents one respiratory disease and each area indicates inference scores between genes and diseases

Fig. 6

Functional prediction of miRNA targeting hub genes. a. miRNA-mRNA network. The red represents mRNA of hub genes, green represents predicted miRNA. b-f. Bubble chart shows the KEGG pathway enrichment of each mRNA’s predicted miRNAs. The sizes of the dots indicate the number of genes and colors of dots indicate p-value