CircRNA expression profiles and circRNA-miRNA-mRNA crosstalk in allergic rhinitis

Abstract

Background: Circular RNAs (circRNAs) are involved in inflammation; however, their role in allergic rhinitis (AR) remains unclear. In this study, we analyzed circRNA expression and identified a circRNA-miRNA-mRNA network through which circRNAs regulate AR pathogenesis.

Methods: We analyzed circRNA, miRNA, and mRNA expression profiles in the nasal mucosa by high-throughput sequencing (HTS), using a fold-change >1.5 and p-value < 0.05 to pinpoint significantly differentially expressed (DE) circRNAs, miRNAs, and mRNAs in AR. A DEcircRNA-DEmiRNA-DEmRNA crosstalk network was then constructed using bioinformatics and statistical analysis. Gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses were performed to identify the biological terms enriched in the network; whereas RT-PCR and Sanger sequencing were used to confirm the circRNAs.

Results: A total of 264 DEcircRNAs were identified by HTS, including 120 upregulated and 144 downregulated in AR compared to controls. A DEcircRNA-DEmiRNA-DEmRNA crosstalk network was constructed with 17 miRNAs, 11 circRNAs, 29 mRNAs, and 64 interaction pairs. These genes were involved in the Wnt signaling pathway, TNF biosynthesis, inflammatory responses, the PI3K-Akt signaling pathway, and Toll-like receptors. Of the 11 DEcircRNAs, hsa_circ_0008668 and circTRIQK were upregulated, whereas hsa_circ_0029853 and circRNA_01002 were downregulated in AR tissues. Sanger sequencing confirmed the back-splicing junctions of these circRNAs.

Conclusions: We constructed a novel DEcircRNA-DEmiRNA-DEmRNA network for AR that provides a basis for future studies to investigate its underlying molecular mechanisms.

Keywords: Allergic rhinitis; Circular RNA; Nasal mucosa; mRNA; microRNA.

Fig. 1

CircRNAs in the nasal mucosa. (A) Box plots showing that circRNA distribution intensities from all normalized datasets were similar between the six samples. (B) A total of 30,936 circRNAs were identified in the nasal mucosa, of which 12,376 were known and 18,560 were novel, by comparing the predicted results with the circBase database (http://www.circbase.org/). (C) Chromosomal circRNA expression distribution. (D) Spliced reads per million (RPM) circRNA distribution. circRNA, circular RNA

Fig. 2

CircRNA, miRNA and mRNA expression. Clustered heat map showing all differentially expressed circRNAs (A), miRNAs (B) and mRNAs (C), respectively (fold-change > 1.5, p < 0.05). Red and blue represent high and low relative expression, respectively. circRNA, circular RNA; miRNA, microRNA

Fig. 3

Top 300 predicted DEcircRNA-miRNA relationships. Some circRNAs were annotated using circBase. Novel circRNAs were described in terms of their genome location. Green and red nodes represent circRNAs and miRNAs, respectively. circRNA, circular RNA; miRNA, microRNA; DE, differentially expressed

Fig. 4

Depiction of the DEcircRNA-DEmRNA network. The network includes 11 circRNAs (green nodes) and 29 mRNAs (yellow nodes). CircRNAs were annotated using circBase. Novel circRNAs were described in terms of their genome location. circRNA, circular RNA; DE, differentially expressed

Fig. 5

Depiction of the DEcircRNA-DEmiRNA-DEmRNA crosstalk network. The network includes 17 miRNAs (red nodes), 11 circRNAs (green nodes), and 29 mRNAs (yellow nodes). CircRNAs and miRNAs were annotated using circBase and miRbase, respectively. Novel circRNAs and miRNAs were described in terms of their genome location. circRNA, circular RNA; miRNA, microRNA; DE, differentially expressed

Fig. 6

Bioinformatics analysis of the DEcircRNA-DEmiRNA-DEmRNA crosstalk network. (A) GO enrichment for biological processes, cell components, and molecular functions, respectively. (B) KEGG pathway analysis of the involvement of linear transcripts in different biological pathways. Bar plot depicting the enrichment score of significantly enriched pathways. circRNA, circular RNA; miRNA, microRNA; DE, differentially expressed; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes

Fig. 7

Sanger sequencing and RT-PCR of circRNAs. (A–D) Back-splice junction sequences for hsa_circ_0008668, hsa_circ_0029853, circTRIQK, and circRNA_01002 were confirmed by Sanger sequencing, respectively. Arrows represent divergent primers binding to the genomic region of circRNAs. (E–H) RT-PCR results for circRNAs in the nasal mucosa of ten patients with AR and ten control subjects. Hsa_circ_0008668 (E) and circTRIQK (G) were upregulated in AR. whereas hsa_circ_0029853 (F) and circRNA_01002 (H) were downregulated, consistent with the HTS data. Data represent the mean ± SD of at least three independent experiments.∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. circRNA, circular RNA