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. 2022 Jul 7:13:917454.
doi: 10.3389/fgene.2022.917454. eCollection 2022.

Identification of circRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathway Axis Involved in Tetralogy of Fallot

Zunqi Kan  1 , Wenli Yan  1 , Ning Wang  1 , Yuqing Fang  1 , Huanyu Gao  1 , Yongmei Song  2
Affiliations

Identification of circRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathway Axis Involved in Tetralogy of Fallot

Zunqi Kan et al. Front Genet. .

Abstract

Tetralogy of Fallot (TOF) is one of the most common cyanotic congenital heart diseases (CHD) worldwide; however, its pathogenesis remains unclear. Recent studies have shown that circular RNAs (circRNAs) act as "sponges" for microRNAs (miRNAs) to compete for endogenous RNA (ceRNA) and play important roles in regulating gene transcription and biological processes. However, the mechanism of ceRNA in TOF remains unclear. To explore the crucial regulatory connections and pathways of TOF, we obtained the human TOF gene, miRNA, and circRNA expression profiling datasets from the Gene Expression Omnibus (GEO) database. After data pretreatment, differentially expressed mRNAs (DEmRNAs), microRNAs (DEmiRNAs), and circRNAs (DEcircRNAs) were identified between the TOF and healthy groups, and a global triple ceRNA regulatory network, including circRNAs, miRNAs, and mRNAs based on the integrated data, was constructed. A functional enrichment analysis was performed on the Metascape website to explore the biological functions of the selected genes. Then, we constructed a protein-protein interaction (PPI) network and identified seven hub genes using the cytoHubba and MCODE plug-ins in the Cytoscape software, including BCL2L11, PIK3R1, SOCS3, OSMR, STAT3, RUNX3, and IL6R. Additionally, a circRNA-miRNA-hub gene subnetwork was established, and its enrichment analysis results indicated that the extrinsic apoptotic signaling pathway, JAK-STAT signaling pathway and PI3K-Akt signaling pathway may be involved in the pathogenesis of TOF. We further identified the hsa_circ_000601/hsa-miR-148a/BCL2L11 axis as a crucial signaling pathway axis from the subnetwork. This study provides a novel regulatory network for the pathogenesis of TOF, revealing the possible molecular mechanisms and crucial regulatory pathways that may provide new strategies for candidate diagnostic biomarkers or potential therapeutic targets for TOF.

Keywords: circular RNA; competing endogenous RNA; congenital heart disease; regulatory networks; tetralogy of fallot.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Flowchart of the present study to construct a circRNA-miRNA-mRNA regulatory network and identify crucial pathway axis of TOF.
FIGURE 2
FIGURE 2
Acquire differentially expressed genes of TOF (DEmRNAs). (A) Volcano plots for DEmRNAs, the red and green points represent up and down expressed DEmRNAs respectively. (B) A heatmap for 40 DEmRNAs we selected, the change in color represents the difference in expression.
FIGURE 3
FIGURE 3
Acquire differentially expressed microRNAs (DEmiRNAs) of TOF. (A) Volcano plots for DEmiRNAs, the red and green points represent up and down expressed DEmiRNAs respectively. (B) A heatmap for 40 DEmiRNAs we selected, the change in color represents the difference in expression.
FIGURE 4
FIGURE 4
Acquire differentially expressed circRNAs (DEcircRNA) of TOF. (A) Volcano plots for DEcircRNAs, the red and green points represent up and down expressed DEcircRNAs respectively. (B) A heatmap for 40 DEcircRNAs we selected, the change in color represents the difference in expression.
FIGURE 5
FIGURE 5
Veen plot of mRNAs predicted by DEmiRNAs.
FIGURE 6
FIGURE 6
The view of DEcircRNA-DEmiRNA-DEmRNA regulatory network. The network includes 29 miRNAs, 13 circRNAs, 88 mRNAs and 231 edges. The yellow diamonds represented mRNA, the blue V shape represented miRNA, and the red circles represented circRNA.
FIGURE 7
FIGURE 7
GO and KEGG functional enrichment analyses of mRNAs in the ceRNA regulatory network. (A) Biological process analysis. (B) Cellular component analysis. (C) Molecular function analysis. The color intensity of the nodes shows how rich the analysis is. The enrichment factor is defined as the ratio of differential genes in the whole genome. The size of dot represents the number of genes in the pathway. (D) KEGG pathway analysis. The green rectangle represented the pathway and the yellow diamonds represented mRNA.
FIGURE 8
FIGURE 8
Construction of PPI network and identification of hub genes. (A) The PPI network of 88 mRNAs was selected by crossover. Red indicates upregulation, blue indicates downregulation, and color depth indicates upregulation degree. The size of the circle indicates the size of the degree value. (B) Key module of the PPI network. (C–E) The hub genes were identified using four models (Degree, MCC, MNC, and Degree). (F) Venn diagram was used to identify the 7 hub genes in TOF.
FIGURE 9
FIGURE 9
Significantly enriched items GO analysis and KEGG pathway analysis of hub genes.
FIGURE 10
FIGURE 10
The circRNA-miRNA-hub gene subnetwork and crucial signaling pathway axis. (A) The Sankey diagram of the subnetwork in TOF. Each rectangle represents an element (circRNA, miRNA, mRNA), and the size of the rectangle indicates the degree of connection of each component. (B) Identification of crucial signaling pathway axis. Red indicates up-regulation, blue indicates down-regulation, and color depth indicates up-regulation degree. (C) Predicted the structure of hsa_circ_000601 based on the CircPrimer software.
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