Identification of Potential Immune-Related circRNA-miRNA-mRNA Regulatory Network in Intestine of Paralichthys olivaceus During Edwardsiella tarda Infection.

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 ß-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.

Molecular identification and expression analysis of four Lysin motif (LysM) domain-containing proteins from turbot (Scophthalmus maximus).

Lysin motif (LysM) is involved in chitin, peptidoglycan and other structurally-related oligosaccharides recognition and binding, and it is important for the biological processes of responsing to bacterial and viral infections and pathogen defense. LysM is also a widely spread protein, ranging from prokaryotes to eukaryotes, including bacteria, plants and mammals. However, research of LysM in teleosts especially in marine fish was rarely scarce. In the present study, four novel LysM domain-containing proteins in turbot (Scophthalmus maximus), named as SmLysMd1, SmLysMd2, SmLysMd3, and SmLysMd4, were cloned and identified firstly. The full-length cDNA of SmLysMd1 was 1235 bp with a 678 bp ORF, capable of encoding a peptide of 225 amino acids. The complete cDNA sequence of SmLysMd2 was 1273 bp, and contained a 675 bp ORF, encoding a predicted protein of 224 amino acids. The full-length of SmLysMd3 cDNA was 2132 bp, containing a ORF of 987 bp, with a ORF of encoding 328 amino acids. The full-length SmLysMd4 cDNA was 1115 bp contained a 888 bp ORF, encoding 295 amino acids. And all the four predicated proteins contained a specific LYSM domain. Moreover, SmLysMd1 and SmLysMd2 belong to the intracellular non-secretory types, and SmLysMd3 and SmLysMd4 belong to the anchored transmembrane types. In addition, the four SmLysMd were ubiquitously expressed in all the examined tissues. Moreover, the SmLysMds levels were up-regulated in muscle and liver, and had a reduce tendency immediately in different degree following Vibrio vulnificus challenge, indicating that the turbot LysM could be participant in the immune responses to bacterial infections. The present result of LysM in turbot for the first time in a marine fish will provide foundation knowledge for the functions studies of LysM in immune responses. Further studies should be carried out to better understand their immune mechanism in turbot and other teleosts.

Molecular identification and function analysis of bactericidal permeability-increasing protein/LPS-binding protein 1 (BPI/LBP1) from turbot (Scophthalmus maximus).

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) play important roles in host antimicrobial defense. In the present study, we identified one isoform of BPI/LBP gene from turbot (Scophthalmus maximus), designated as SmBPI/LBP1. The full-length cDNA sequence of SmBPI/LBP1 was 1826 bp, which encoding one secreted protein with 480 amino acid residues. Structurally, the SmBPI/LBP1 showed high similarity to its homologs from other vertebrates or invertebrates, which all contained a signal peptide, a BPI/LBP/CETP N-terminal with a LPS-binding domain, and a BPI/LBP/CETP C-terminal domain. The deduced amino acid sequences of SmBPI/LBP1 shared significant similarity to BPI/LBP of Seriola lalandi dorsalis (71%) and Paralichthys olivaceus (69%). Phylogentic analysis further supported that SmBPI/LBP1 act as a new member of vertebrate BPI/LBP family. SmBPI/LBP1 was ubiquitously expressed in all tested tissues, with the highest expression level in spleen tissue. The mRNA expression of SmBPI/LBP1 in spleen and kidney were significantly up-regulated after Vibrio vulnificus challenge. Finally, the recombinant SmBPI/LBP1 showed high affinity to lipopolysaccharide, followed by peptidoglycan and lipoteichoic acid, which is the ubiquitous component of Gram-negative or Gram-positive bacteria. These results indicated that SmBPI/LBP1 probably played important roles in immune response against bacteria infection.