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Circular RNAs (circRNAs) were recently recognized to act as competing endogenous RNAs and play roles in gene expression regulation. Previous studies in humans and silkworms have shown that circRNAs take part in immune regulation. Here, we conducted coelomocyte circRNA sequencing to explore its immune functions in healthy and skin ulceration syndrome (SUS)-diseased sea cucumbers. A total of 3,592 circRNAs were identified in libraries with diversified circularization patterns compared with animal models. The common intron-pairing-driven circularization models are not popular in sea cucumber genome, which was replaced with intergenic region circularization. The accuracy of these identified circRNAs was further validated by Sanger sequencing and RNase R-treated assays. Expression profile analysis indicated that 117 circRNAs were upregulated and 144 circRNAs were downregulated in SUS-diseased condition, of which 71.6% were intergenic-type circRNAs. The interaction network of differentially expressed circRNAs and microRNAs (miRNAs) was constructed and showed that miR-2008 and miR-31, detected with significantly differential expression in SUS-affected samples in a previous study, were predicted to be regulated by 10 and 11 differentially expressed circRNAs with more than 10 binding sites, respectively. Moreover, seven circRNAs were further validated by quantitative real-time PCR, whose variation trends were consistent with circRNA sequencing. All our results supported that intergenic-type circRNAs might have a dominant function in Apostichopus japonicas immune response by acting as miRNA regulators.
FIGURE 1. CircRNAs identified from Apostichopus japonicas coelomocyte. (A) Four types of circRNAs according to their genomic location. (B) Distribution of the number of circRNAs from different types in DC, HC groups and in total.
FIGURE 2. Characterization of circularization in Apostichopus japonicas. (A) Distribution of circRNAs among genes. (B) Number of exons contained within exonic circRNAs. (C) Start and end exons of circRNAs. CircRNAs are most enriched for exons involving positions 2–4. (D) Splice signals of identified circRNAs.
FIGURE 3. GO analysis of the host genes of circRNAs under the categories of BP, CC, and MF.
FIGURE 4. Top 20 enriched KEGG pathways. The size of the circle represents the number of genes. Green to red indicates that the corrected p-value is gradually becoming smaller.
FIGURE 5. Differentially expressed circRNA–miRNA interaction network in the sea cucumber under SUS challenges. Hexagon nodes represent circRNAs, and circle nodes represent miRNAs. The size of the node represents the number of interacted relationships. Gray lines represent more than 10 miRNA binding sites in the circRNA. Green dashed lines represent more than 20 miRNA binding sites in the circRNA. Purple double lines represent more than 30 miRNA binding sites in the circRNA.
FIGURE 6. Interaction network between differentially expressed circRNAs and differentially expressed miRNA in sea cucumber under SUS challenges. Hexagon nodes represent circRNAs, and circle nodes represent miRNAs. Green to red line color represents the number of miRNA binding sites in the circRNA from 5 to 32.
FIGURE 7. Validation of circRNAs by RT-PCR and Sanger sequencing. Red arrows represent back-splicing sites.
FIGURE 8. Experimental validation of circRNAs. (A) qRT-PCR analysis of seven circRNAs with different expression patterns in response to SUS. β-actin is the internal reference. N = 3, ∗p < 0.05, ∗∗p < 0.01. (B) Outward-facing primers and inward-facing primers were designed to compare total and RNase R-treated RNAs from coelomocytes by gel electrophoresis. Linear mRNA forms are depleted by RNase R, whereas circular ones are resistant. CircRNA: AJAPscaffold1149:136110| 200955; linear RNA: ficolin.
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