Abstract
Olive flounder (Paralichthys olivaceus) is an important economical flatfish in Japan, Korea, and China, but its production has been greatly threatened by disease outbreaks. In this research, we aimed to explore the immune responsive mechanism of P. olivaceus against Edwardsiella tarda infection by profiling the expression of circRNA, miRNA, and mRNA by RNA-seq and constructing a regulatory circular circRNA–miRNA–mRNA network. Illumina sequencing of samples from normal control (H0), 2 h (H2), 8 h (H8), and 12 h (H12) post-challenge was conducted. Differentially expressed (DE) circRNA (DE–circRNAs), miRNAs (DE–miRNAs), and mRNAs [differential expression genes (DEGs)] between challenge and control groups were identified, resulting in a total of 62 DE–circRNAs, 39 DE–miRNAs, and 3,011 DEGs. Based on the differentially expressed gene results, miRNA target interactions (circRNA–miRNA pairs and miRNA–mRNA pairs) were predicted by MiRanda software. Once these paired were combined, a preliminary circRNA–miRNA–mRNA network was generated with 198 circRNA–miRNA edges and 3,873 miRNA–mRNA edges, including 44 DE–circRNAs, 32 DE–miRNAs, and 1,774 DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to evaluate the function of the DEGs in this network, and we focused and identified two important intestinal immune pathways (herpes simplex infection and intestinal immune network for IgA production) that showed statistical significance between the challenge and control groups. Furthermore, three critical DEGs (nectin2, MHC II α-chain, and MHC II β-chain) were identified, mapped into the preliminary circRNA–miRNA–mRNA network, and new circRNA–miRNA–mRNA regulatory networks were constructed. In conclusion, we, for the first time, identified circRNA–miRNA–mRNA network from P. olivaceus in the pathogenesis of E. tarda and provided valuable resources for further analyses of the molecular mechanisms and signaling networks.
Highlights
Circular RNAs, identified from RNA viruses in the 1970s (Sanger et al, 1976), were initially treated as viral genomes or by-products of rare mis-splicing, and they have long been thought to be nonfunctional (Capel et al, 1993)
The results showed that E. tarda could 1) up-regulate mRNA levels of intestinal pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, IL-8, IL-16, and IL-17D, tumor necrosis factor α (TNF-α), and granulocyte colonystimulating factor (G-CSF); and 2) down-regulate the mRNA levels of anti-inflammatory cytokines IL-10 (Figure 2)
Histopathological evaluation and cytokine expression analysis on the posterior intestine of P. olivaceus helped in understanding and determining the pathogenesis of E. tarda
Summary
Circular RNAs (circRNAs), identified from RNA viruses in the 1970s (Sanger et al, 1976), were initially treated as viral genomes or by-products of rare mis-splicing, and they have long been thought to be nonfunctional (Capel et al, 1993). Recent advancements to the high-throughput sequencing technology have benefited large amounts of circRNAs identified in succession from many organisms, such as plants, animals, human beings, fungi, and protists (Li et al, 2018b). Expression profiles and knockout experiments proved that circRNAs have been implicated in neuronal function (Rybak-Wolf et al, 2015; Piwecka and Glazar, 2017) and testes development (Capel et al, 1993; Hansen et al, 2013). Considerable evidences proved that circRNAs are not accidental by-products but represent an essential part of noncoding RNA families
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