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. 2021 Jun 21:9:685741.
doi: 10.3389/fcell.2021.685741. eCollection 2021.

Comprehensive Analysis of Peripheral Exosomal circRNAs in Large Artery Atherosclerotic Stroke

Qi Xiao  1 , Ruihua Yin  1 , Yuan Wang  1 , Shaonan Yang  1 , Aijun Ma  1 , Xudong Pan  1   2 , Xiaoyan Zhu  3
Affiliations

Comprehensive Analysis of Peripheral Exosomal circRNAs in Large Artery Atherosclerotic Stroke

Qi Xiao et al. Front Cell Dev Biol. .

Abstract

Exosomes are crucial vehicles in intercellular communication. Circular RNAs (circRNAs), novel endogenous noncoding RNAs, play diverse roles in ischemic stroke. Recently, the abundance and stability of circRNAs in exosomes have been identified. However, a comprehensive analysis of exosomal circRNAs in large artery atherosclerotic (LAA) stroke has not yet been reported. We performed RNA sequencing (RNA-Seq) to comprehensively identify differentially expressed (DE) exosomal circRNAs in five paired LAA and normal controls. Further, quantitative real-time PCR (qRT-PCR) was used to verify the RNA-Seq results in a cohort of stroke patients (32 versus 32). RNA-Seq identified a total of 462 circRNAs in peripheral exosomes; there were 25 DE circRNAs among them. Additionally, circRNA competing endogenous RNA (ceRNA) network and translatable analysis revealed the potential functions of the exosomal circRNAs in LAA progression. Two ceRNA pathways involving 5 circRNAs, 2 miRNAs, and 3 mRNAs were confirmed by qRT-PCR. In the validation cohort, receiver operating characteristic (ROC) curve analysis identified two circRNAs as possible novel biomarkers, and a logistic model combining two and four circRNAs increased the area under the curve compared with the individual circRNAs. Here, we show for the first time the comprehensive expression of exosomal circRNAs, which displayed the potential diagnostic and biological function in LAA stroke.

Keywords: RNA sequencing; ceRNA network; circular RNAs; exosomes; large artery atherosclerotic stroke.

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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
Characteristics of exosomes isolated from peripheral plasma. (A) Representative TEM micrograph of exosomes (scale bar of 100 nm). (B) NTA analysis of exosomes enriched from plasma indicated they were approximately 50–150 nm in diameter. (C) Western blotting showing the protein expression of three positive protein markers (CD9, CD63, and TSG101) and a negative protein marker (GRP94) of exosomes.
FIGURE 2
FIGURE 2
Expression profiles of exosome-circRNAs from RNA-Seq. (A) Various RNA biotypes expressed as a percentage of the total sequencing reads by taking the average reads of each biotype across each group. (B) circRNAs from exons, introns, or intergenic regions. (C) The lengths of circRNAs in each subject. (D) Volcano plots of differentially expressed circRNAs. The red and green dots indicate upregulated and downregulated circRNAs, respectively. (E) Heat maps of differentially expressed circRNAs.
FIGURE 3
FIGURE 3
Functional enrichment of the source genes of the DE circRNAs. (A) Top 20 biological processes, cell components, and molecular functions according to GO analysis. (B) Top 20 pathways according to KEGG analysis visualized on a bulb map. The color of the dot corresponds to different p-value ranges, and the size of the dot indicates the number of genes in the pathway.
FIGURE 4
FIGURE 4
Differential expression and hierarchical cluster analysis of miRNAs, and mRNAs. Volcano plots of significantly differentially expressed RNAs. The red and green dots indicate upregulated and downregulated genes, respectively. The horizontal dotted lines represent a p-value of 0.05, and the vertical dotted lines represent a fold change of 1.5-fold. Heat maps of the RNA expression hierarchical patterns. (A,B) Volcano plots of differentially expressed miRNAs, mRNAs. (C,D) Heat maps of differentially expressed miRNAs, mRNAs.
FIGURE 5
FIGURE 5
Protein–Protein Interaction and circRNA-miRNA-mRNA network. (A) PPI Networks of DE exosomal mRNAs. (B) Exosomal miRNA-mRNA regulatory networks. (C) Exosomal circRNA-miRNA regulatory networks. (D) circRNA-miRNA-mRNA regulatory network. The yellow triangles represent miRNAs, red nodes circRNAs, and blue frames mRNAs.
FIGURE 6
FIGURE 6
Representative circRNA-miRNA-mRNA network expression validation by qRT-PCR. (A) Representative circRNA-miRNA-mRNA network. (B–G) The fold changes of LAA and normal subjects’ expression values of hsa_circ_0000698, hsa_circ_0002775, hsa_circ_0005585, hsa_circ_0043837, hsa-miR-16, and VWF. (H–K) The fold changes of the LAA and normal subjects’ expression values of novel_circ_0010155, has-miR-393, septin9, and MYLK2.
FIGURE 7
FIGURE 7
The exosomal circRNAs IRES scores and translation mode pattern. (A) Percentage distribution of circRNA IRES prediction scores. (B) The Exosomal circRNAs translation mode pattern.
FIGURE 8
FIGURE 8
Performance of exosomal circRNAs according to ROC curve analysis. ROC curves of exosome-derived (A) novel_circ_0010155, (B) hsa_circ_0005585, (C) the four circRNA model, and (D) novel_circ_001015 plus hsa_circ_0005585.
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