Genome-wide characterization of integrin (ITG) gene family and their expression profiling in half-smooth tongue sole (Cynoglossus semilaevis) upon Vibrio anguillarum infection.

Integrins (ITGs) are transmembrane heterodimer receptors with ITGα subunit and ITGß subunit, participating in various physiological processes, including immunity. At present, systematic research on ITGs in teleost is scarce, especially in half-smooth tongue sole (Cynoglossus semilaevis). In this study, a set of 28 ITG genes in half-smooth tongue sole have been identified and characterized. The phylogenetic analysis showed that ITGα and ITGß subunits were respectively classified into five and two clusters, consistent with previous studies. The selection pressure analysis indicated that most of ITG genes were under purifying selection, except for ITGα11b and ITGαL with positive selection. The expression profiles of eight selected ITG genes, including ITGα1, ITGα5, ITGα8, ITGα11, ITGß1, ITGß2, ITGß3, and ITGß8, were analyzed in healthy tissues and after infection with Vibrio anguillarum, revealed their implications in immune response. The study provided a comprehensive characterization and expression analysis of ITG genes in half-smooth tongue sole, setting a solid foundation for further functional studies and promising potential in disease control.

Immune role of the complement component 6 gene and its associated novel miRNA, miR-727, in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 6 (C6) gene is a component of the membrane attack complex (MAC), which causes rapid lytic destruction of bacteria. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene stability, including that of immune genes. However, current research on the function of C6 and its regulation by miRNAs is lacking. In the present study, we identified and characterized C6 and a novel miRNA, miR-727 (designated CsC6 and Cse-miR-727, respectively), of the half-smooth tongue sole (Cynoglossus semilaevis) that responded to infection with Vibrio anguillarum, a Gram-negative pathogen of marine fish. The full-length cDNA of CsC6 contained a 256 bp 5' untranslated region (5'-UTR), a 2820 bp open reading frame (ORF) encoding 939 amino acids, and a 205 bp 3'-UTR. SMART analysis showed that CsC6 contains typical C6 domains, including three TSP1 domains, one LDLa domain, one MACPF domain, two CCP domains and two FIMAC domains. CsC6 and Cse-miR-727 are widely expressed in the 13 tissues of half-smooth tongue sole, and their expression in immune tissues is significantly changed after V. anguillarum infection, generally showing an inverse trend. We confirmed that CsC6 was the target gene of Cse-miR-727 using the dual luciferase reporter assay and that Cse-miR-727 regulated CsC6 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The hepatic expression levels of not only the MAC components C7, C8α, C8ß, C8γ and C9 but also the MAPKs, NF-κß, AP-1, IL1ß, IL6 and TNFα, which are involved in many signaling pathways, changed significantly in half-smooth tongue sole following stimulation with the Cse-miR-727 agomir and inhibitor. This evidence suggested that CsC6 could be mediated by Cse-miR-727 to affect MAC assembly and immune signaling pathways in half-smooth tongue soles. To our best knowledge, this study is the first to investigate the regulatory mechanism and immune response of complement genes mediated by miRNAs in fish.

Combining of transcriptomic and proteomic data to mine immune-related genes and proteins in the liver of Cynoglossus semilaevis challenged with Vibrio anguillarum.

The liver is a multi-functional organ including metabolism, substance synthesis, detoxification, and various immune functions, and its role in immunity has attracted more and more attention. However, research on the liver immune response of fish infected by pathogenic bacteria is currently lacking. In this study, the transcriptomics and proteomics of the liver of Cynoglossus semilaevis infected with Vibrio anguillarum were analyzed. A total of 1470 genes and 497 proteins were differentially expressed in the pairwise comparison of obvious symptoms of infection (HOSG), no obvious symptoms of infection (NOSG) and PBS treatment (CG). Gene ontology and KEGG enrichment pathways analysis showed that differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were mainly enriched in toll-like receptors (TLRs), complement and coagulation cascades, nucleotide oligomerization domain (NOD)-like receptors (NLRs), mitogen-activated protein kinase (MAPK) and phagosome signaling pathways, which suggested the combined action of the five pathways were significant to enhance the liver immune defense. The combination of transcriptomic and proteomic analysis showed that ITGß1, C3, C5 and MRC1 were significantly up-regulated, which might play an important role in the liver immune response to the recognition of V. anguillarum, inflammatory response and phagocytosis. The transcriptome and proteome data we obtained provide information on some key genes and proteins for further study of the mechanism of liver immune response.

Analysis of lncRNA and mRNA expression profiles in peripheral blood leukocytes of the half-smooth tongue sole (Cynoglossus semilaevis) treated with chitosan oligosaccharide.

Long noncoding RNAs (lncRNAs) play a multifaceted role in transcriptional regulation and are important regulators of immune function. Scarce information is available regarding lncRNAs in fish. Peripheral blood mononuclear cells participate in the immune response of fish and aid resistance to infection with pathogenic microorganisms. Chitosan oligosaccharide can improve cellular and humoral immunity to enhance disease resistance in fish. In this study, we obtained peripheral blood leukocytes from half-smooth tongue sole and studied the effect of chitosan oligosaccharide on the lncRNA-mRNA expression profile of these cells using high-throughput sequencing and bioinformatics techniques. A total of 609 differentially expressed mRNAs and 50 differentially expressed lncRNAs were identified. The GO term enrichment analysis of the differentially expressed genes was annotated by 220 GO terms, 137 biological processes, 18 cellular components, and 65 molecular functions. Sixteen KEGG pathways, including immune signaling pathways, metabolism, and genetic information processing, were significantly enriched in differentially expressed genes. Thirty-six differentially expressed lncRNAs and 32 differentially expressed mRNAs produced a coexpression network containing 90 relationship pairs. The prediction of lncRNA target genes revealed 244 lncRNAs that potentially cis-regulated 294 differentially expressed mRNAs. qPCR verified that the expression levels of 17 differentially expressed lncRNAs and 15 differentially expressed mRNAs were consistent with the RNA-Seq results. Among them, 6 lncRNAs and 7 mRNAs were differentially expressed genes obtained from the prediction and analysis of lncRNA target genes, and 8 lncRNAs and 4 mRNAs were differentially expressed genes that participated in the construction of the coexpression network. In peripheral blood leukocytes after chitosan oligosaccharide treatment, as well as in peripheral blood and spleen after Vibrio anguillarum stimulation, lncRNAs and mRNAs showed significant differential expression. The results indicated that they may be related to the immune response, providing novel reference information for further research on the role of lncRNAs in immune regulation in half-smooth tongue sole.