Construction of the circRNA-miRNA-mRNA Regulatory Network of an Abdominal Aortic Aneurysm to Explore Its Potential Pathogenesis

Abstract

Background: Abdominal aortic aneurysm (AAA) is a progressive cardiovascular disease, which is a permanent and localized dilatation of the abdominal aorta with potentially fatal consequence of aortic rupture. Dysregulation of circRNAs is correlated with the development of various pathological events in cardiovascular diseases. However, the function of circRNAs in abdominal aortic aneurysm (AAA) is unknown and remains to be explored. This study is aimed at determining the regulatory mechanisms of circRNAs in AAAs. This study was aimed at exploring the underlying molecular mechanisms of abdominal aortic aneurysms based on the competing endogenous RNA (ceRNA) regulatory hypothesis of circRNA, miRNA, and mRNA.

Methods: The expression profiles of circRNAs (GSE144431), miRNAs (GSE62179), and mRNAs (GSE7084, GSE57691, and GSE47472) in human tissue sample from the aneurysm group and normal group were obtained from the Gene Expression Omnibus database, respectively. The circRNA-miRNA-mRNA network was constructed by using Cytoscape 3.7.2 software; then, the protein-protein interaction (PPI) network was constructed by using the STRING database, and the hub genes were identified by using the cytoHubba plug-in. The circRNA-miRNA-hub gene regulatory subnetwork was formed to understand the regulatory axis of hub genes in AAAs.

Results: The present study identified 40 differentially expressed circRNAs (DECs) in the GSE144431, 90 differentially expressed miRNAs (DEmiRs) in the GSE62179, and 168 differentially expressed mRNAs (DEGs) with the same direction regulation (130 downregulated and 38 upregulated) in the GSE7084, GSE57691, and GSE47472 datasets identified regarding AAAs. The miRNA response elements (MREs) of three DECs were then predicted. Four overlapping miRNAs were obtained by intersecting the predicted miRNA and DEmiRs. Then, 17 overlapping mRNAs were obtained by intersecting the predicted target mRNAs of 4 miRNAs with 168 DEGs. Furthermore, the circRNA-miRNA-mRNA network was constructed through 3 circRNAs, 4 miRNAs, and 17 mRNAs, and three hub genes (SOD2, CCR7, and PGRMC1) were identified. Simultaneously, functional enrichment and pathway analysis were performed within genes in the circRNA-miRNA-mRNA network. Three of them (SOD2, CCR7, and PGRMC1) were suggested to be crucial based on functional enrichment, protein-protein interaction, and ceRNA network analysis. Furthermore, the expression of SOD2 and CCR7 may be regulated by hsa_circ_0011449/hsa_circ_0081968/hsa-let-7f-5p; the expression of PGRMC1 may be regulated by hsa_circ_0011449/hsa_circ_0081968-hsa-let-7f-5p/hsa-let-7e-5p.

Conclusion: In conclusion, the ceRNA interaction axis we identified may be an important target for the treatment of abdominal aortic aneurysms. This study provided further understanding of the potential pathogenesis from the perspective of the circRNA-related competitive endogenous RNA network in AAAs.

Figure 1

Flow diagram of data processing. The difference expression of three datasets (circRNA dataset, miRNA dataset, and mRNA dataset) was analyzed, and then, the intersection was selected; construction of the ceRNA network, protein-protein interaction network and functional enrichment analysis, and finally determination of the key genes.

Figure 2

Hierarchical clustering and heat map analysis of differentially expressed circRNAs (a) and mRNAs (b–d). (a) GSE144431, circRNA; (b) GSE7084, mRNA; (c) GSE57691, mRNA; (d) GSE47472, mRNA. The green color indicates low expression, the red indicates high expression. Column: an aortic sample. Differentially expressed circRNA molecules were screened under the cut-off criteria ∣log2FC | >1 and the P value (P < 0.1). Differentially expressed mRNA molecules were screened under the cut-off criteria ∣log2FC | >2 and the P value (P < 0.05).

Figure 3

Volcano plots of differentially expressed circRNAs (a) and mRNAs (b–d). (a) GSE144431, circRNA; (b) GSE7084, mRNA; (c) GSE57691, mRNA; (d) GSE47472, mRNA. Green spots: downexpressed RNA molecules; red spots: upexpressed RNA molecules. Differentially expressed circRNA molecules were screened under the cut-off criteria ∣log2FC | >1 and the P value (P < 0.1). Differentially expressed mRNA molecules were screened under the cut-off criteria ∣log2FC | >2 and the P value (P < 0.05).

Figure 4

Venn diagram analysis and GO/KEGG analysis of 168 DEG coregulation modes in the three datasets: (a) intersection of downregulated genes (GSE7084, GSE57691, and GSE47472); (b) intersection of upregulated genes (GSE7084, GSE57691, and GSE47472); (c) bubble chart and barplot of 168 DEGs by GO analysis; (d) bubble chart and barplot of 168 DEGs by KEGG analysis.

Figure 5

Protein and protein interaction network for DEG coregulation modes. Dots represent gene expression, and lines represent interaction relationships. Blue: downregulated; red: upregulated.

Figure 6

The ceRNA interaction network of circRNA-miRNA-mRNA. Round represents mRNAs, square shapes represent miRNAs, and triangles represent circRNAs. Connection represents interaction.

Figure 7

Differential expression analysis of hub genes from PPI network and ceRNA network genes. (a) Hub gene relationship networks from the PPI network. (b) Venn diagram analysis of hub genes from PPI network and ceRNA network genes. (c) Box plot of the differentially expressed gene CCR7 in the three datasets. (d) Box plot of the differentially expressed gene PGRMC1 in the three datasets. (e) Box plot of the differentially expressed gene SOD2 in the three datasets. Green: normal samples; purple: aneurysm samples; n: number of samples.