Comprehensive analysis of circRNA-miRNA-mRNA network related to angiogenesis in recurrent implantation failure

Abstract

Background: Abnormal endometrial blood flow causes a decrease in endometrial receptivity and is considered a relatively independent risk factor for recurrent implantation failure (RIF). This study aimed to explore the potentially functional circRNA-miRNA-mRNA network in RIF, and further explore its mechanism.

Methods: Datasets were downloaded from the GEO database to identify differentially expressed circRNAs, miRNAs and mRNAs. The circRNA-miRNA-mRNA and PPI networks were constructed using Cytoscape 3.6.0 and the STRING database, the hub genes were identified with the cytoHubba plug-in, and a circRNA-miRNA-hub mRNA regulatory sub-network was constructed. Then, GO and KEGG pathway enrichment analyses of the hub genes were performed to comprehensively analyze the mechanism of hub mRNAs in RIF. Due to the results of circRNAs-miRNAs-hub mRNAs regulatory network, we verified the expression of circRNA_0001721, circRNA_0000714, miR-17-5p, miR-29b-3p, HIF1A and VEGFA in the RIF mouse model by qRT‒PCR and western blotting.

Results: We initially identified 175 DEmRNAs, 48 DEmiRNAs and 56 DEcircRNAs in RIF associated with angiogenesis and constructed a circRNA-miRNA‒mRNA network and PPI network. We further identified six hub genes in the acquired network. Based on these genes, functional enrichment analysis revealed that the HIF-1 signaling pathway plays a vital role in endometrial angiogenesis in RIF. In addition, the interaction networks of circRNA_0001721/miR-17-5p/HIF1A and the circRNA_0000714/miR-29b-3p/VEGFA axis were predicted. In the RIF mouse model, circRNA_0001721, circRNA_0000714, HIF1A and VEGFA were down-regulated, whereas miR-17-5p and miR-29b-3p were up-regulated according to qRT‒PCR and western blotting.

Conclusion: This study revealed that the HIF-1 signaling pathway plays a vital role in endometrial angiogenesis in RIF. The circRNA_0001721/miR-17-5p/HIF1A and circRNA_0000714/miR-29b-3p/VEGFA axes might play a role in the pathogenesis of endometrial angiogenesis in RIF.

Keywords: Angiogenesis; Circular RNA; Endometrial receptivity; Molecular pathogenesis; Recurrent implantation failure; Regulatory network; Signaling pathway.

Fig. 1

Study design flowchart. circRNA, circular RNA; miRNA, microRNA; RIF, recurrent implantation failure. DEcircRNAs, differentially expressed circRNAs; DEmiRNAs, differentially expressed miRNAs; DEmRNAs, differentially expressed mRNAs

Fig. 2

Boxplots, volcano plots and Venn diagram of mRNAs for GSE103465. (A) Boxplot of GSE103465. (B) Volcano plots of DEmRNAs based on GSE103465. (C) Venn diagram of mRNAs for GeneCards and GSE103465, where the intersection section is predicted DEmRNAs asscociated with angiogenesis in RIF. circRNA, circular RNA; miRNA, microRNA; RIF, recurrent implantation failure

Fig. 3

Boxplots, volcano plots and Venn diagram of miRNAs for GSE121219. (A) Boxplot of GSE121219. (B) Volcano plots of DEmiRNAs based on GSE121219. (C) Venn diagram of miRNAs for miRDIP and GSE121219, where the intersection section is predicted DEmiRNAs asscociated with angiogenesis in RIF. circRNA, circular RNA; miRNA, microRNA; RIF, recurrent implantation failure

Fig. 4

Boxplots, volcano plots and Venn diagram of circRNAs for GSE147442. (A) Boxplot of GSE147442 after standardization. (B) Volcano plots of DEcircRNAs based on GSE147442. (C) Venn diagram of miRNAs for starBase 2.0 and GSE147442, where the intersection section is predicted DEcircRNAs asscociated with angiogenesis in RIF. circRNA, circular RNA; miRNA, microRNA; RIF, recurrent implantation failure

Fig. 5

Figure 5 CircRNA-miRNA-mRNA regulatory network, which consists of 8 DEcircRNAs, 10 DEmiRNAs and 45 DERNAs. DEcircRNA, differentially expressed circular RNA; DEmiRNA, differentially expressed micro RNA; DERNA, differentially expressed RNA. Arrow-shape nodes: circRNAs, triangle nodes: miRNAs, ellipse-shaped nodes: mRNAs

Fig. 6

A PPI network and circRNA–miRNA–hub gene regulatory subnetwork. (A) A PPI network of the fifty-five target genes associated with angiogenesis in RIF. (B) Six hub genes extracted by cytoHubba plug-in. (C) CircRNA–miRNA–hub gene regulatory subnetwork, consisting of 3 circRNAs, 3 miRNAs, and 3 mRNAs. PPI, protein–protein interaction; circRNA, circular RNA; miRNA, microRNA; RIF, recurrent implantation failure

Fig. 7

GO function analysis histogram. BP is marked by dark cyan, CC is marked by sienna and MF is marked by steel blue. The bar chart was constructed through the bioinformatics platform

Fig. 8

qRT-PCR validation. (A) The expression levels of CircRNA_0001721and circRNA_0000714. (B) The expression levels of miR-17-5p and miR-29b-3p. (C) The expression levels of HIF1A and VEGFA

Fig. 9

Western blot validation. (A) The relative levels of VEGFA. (B) The relative levels of HIF1A. (C) western blot image of HIF1A and VEGF. Full-length blots and gels are presented in Supplementary Figure and the grouping of gels/blots cropped from different gels