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MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8, were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are small non-coding RNAs involved in various biological processes, including immunity. Previously, we investigated the miRNAs of half-smooth tongue sole (Cynoglossus semilaevis) and found that miR-722 (designated Cse-miR-722) was significantly differentially expressed after infection with Vibrio anguillarum, reflecting its importance in immune response. Our preliminary bioinformatic analysis suggested that Cse-miR-722 could target C5aR1 (designated CsC5aR1), which was known to play crucial roles in complement activation and inflammatory response, as a receptor of C5a. However, the underlying mechanisms of their interactions and specific functions in inflammatory and immune response are still enigmas. In this study, we successfully cloned the precursor sequence of Cse-miR-722 (94 bp) and the full length of CsC5aR1 (1541 bp, protein molecular weight 39 kDa). The target gene of Cse-miR-722 was verified as CsC5aR1 by a dual luciferase reporter assay, and Cse-miR-722 was confirmed to regulate CsC5aR1 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The expression of CsC5aR1 and Cse-miR-722 in liver cells and four immune tissues of half-smooth tongue sole changed significantly after LPS stimulation and infection with V. anguillarum. To explore the functional role of Cse-miR-722 in half-smooth tongue sole, we performed both in vitro and in vivo experiments. Cse-miR-722 was observed to affect phagocytosis and respiratory burst activity of macrophages by regulating CsC5aR1 in half-smooth tongue sole. Furthermore, we found that Cse-miR-722 regulated the expression of CsC5aR1, CsC5a, and the inflammatory factors CsIL1-β, CsIL6, CsIL8, and CsTNF-α both in vitro and in vivo. In addition, Cse-miR-722 reduced mortality and pathological damage. This study clarified the regulatory mechanism of Cse-miR-722 on CsC5aR1 and provided insight into the regulatory roles of Cse-miR-722 in immune responses, laying a theoretical foundation for the feasibility of using miR-722 to prevent and control bacterial diseases in teleost., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2023. Published by Elsevier B.V.)

Integrins (ITGs) are transmembrane heterodimer receptors with ITGα subunit and ITGβ 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 ITGβ 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, ITGβ1, ITGβ2, ITGβ3, and ITGβ8, 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

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, C8β, C8γ and C9 but also the MAPKs, NF-κβ, AP-1, IL1β, 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 ITGβ1, 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Collectin 11 (CL-11, also known as collectin kidney-1, CL-K1), a new member of the vertebrate C-type lectin superfamily, plays an important role in innate immunity as a pattern recognition molecule of the lectin complement pathway. However, little is known about CL-11 in teleosts. In the present study, a CL-11 homolog was identified and characterized from half-smooth tongue sole (Cynoglossus semilaevis) (designated as CsCL-11). The full-length cDNA of CsCL-11 is 1220 bp long and includes a 5'untranslated region (5'-UTR) of 180 bp, a 3'-UTR of 218 bp and an open reading frame (ORF) of 819 bp encoding 273 amino acids. Multiple sequence alignment revealed that the deduced CsCL-11 protein has the typical modular architecture (EPN and WTD) conserved throughout vertebrates, suggesting a conserved function of CsCL-11. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed CsCL-11 to be ubiquitously distributed in tissues and highly expressed in the ovary and liver. A pattern of significant upregulation of CsCL-11 expression was observed in the blood, spleen, head kidney and gill at 6 h, 12 h and 24 h after infection with Vibrio anguillarum, and western blotting showed that natural CsCL-11 protein levels in the blood were significantly increased after V. anguillarum infection. Moreover, by binding to various bacteria, recombinant CsCL-11 (rCsCL-11) expressed in HEK-293 T cells displayed strong antibacterial activity. Taken together, these results suggest that CsCL-11 is a unique C-type lectin that is likely involved in host defense against bacterial infection. To our knowledge, this is the first study on CL-11 in marine fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present study, the peripheral blood cells of half smooth tongue sole (Cynoglossus semilaevis) were examined by blood smear under the light microscopy. The proportion of main types of blood cells are as following: erythrocyte occupied the majority (92.3%), followed by thrombocyte (4.15%), granulocyte (1.7%), lymphocyte (1.5%) and monocyte (0.3%), respectively. Meanwhile, the isolation method of monocytes was established, by density gradient centrifugation to isolate mononuclear leukocytes of peripheral blood. In primary culture, the monocytes were adhered to the bottom of the flask without feeder cells and separated easily with suspended leukocytes in the medium in 3 h. After suspended leukocytes were removed, the monocytes multiplied rapidly with the two doubly during the 24 h, then the cells proliferated and kept stable until 48 h. When co-cultured with suspended leukocytes after three days, the monocytes could derive to typical macrophages, of which the size enlarged significantly and showed various forms such as like fried eggs, and giant irregular shape with pseudopod because cells fusion or deformation occurred until macrophages died in about two weeks. Monocytes showed strong respiratory burst activity after treated with Phorbol ester PMA and challenged by bacteria respectively. In addition, macrophage of half smooth tongue sole had typical macrophage features such as phagocytic capability, positive esterase activity, and the considerable expression of M-CSFR, MHC-II, IL-6, IL-10, TNF and arginase genes. That arginase expression in macrophages (3d and 5d after differentiation) was upregulated fluctuant suggest that the cultivation was mixture of alternatively activated type macrophage (M2) in the majority while the classically activated type (M1) win the minority. Furthermore, MHC-Ⅱ, M-CSFR and IL-6 were significantly induced following LPS challenge. Collectively, the present study will be useful for the study on half smooth tongue sole immune systems and immune function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine commercial fish species in China, which suffers from widespread disease outbreaks. Recently, in this regard, our group identified immune-related microRNAs (miRNAs) of C. semilaevis following Vibrio anguillarum infection. Furthermore, miRNA microarray was utilized to characterize the immune roles of important miRNA candidates in response to bacterial infection. Therefore, in the present study, we characterized miR-221 and miR-222 and profiled their expression after challenge. Here, miR-221 and miR-222 precursors were predicted to have a typical hairpin structure. Both miRNAs were expressed in a broad range of tissues in C. semilaevis, while miR-221 and miR-222 were significantly differentially expressed in the immune tissues of C. semilaevis among three small RNA libraries [control group (CG), bacteria-challenged fish without obvious symptoms of infection (NOSG), and bacteria-challenged fish with obvious symptoms of infection (HOSG)]. In order to further characterize and understand the immune response of miR-221 and miR-222, therefore, we profiled miR-221 and miR-222 expression in selected immune tissues after challenge with V. anguillarum. Both miR-221 and miR-222 were upregulated in the liver and spleen, while different expression patterns were observed in the head kidney. In addition, in half-smooth tongue sole head kidney cell line after challenge with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), peptidoglycan (PGN), and red-spotted grouper nervous necrosis virus (RGNNV), both miR-221 and miR-222 showed significant difference in expression response to pathogen. Meanwhile, the target gene of miR-221 and miR-222 was predicted, which indicated that tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 beta (IL-1β) were the target genes of miR-221 and miR-222, respectively. Collectively, these findings indicated that miR-221 and miR-222 have putative roles in innate immune response during C. semilaevis exposure to pathogens. Our findings could expand the knowledge of immune function of C. semilaevis miRNA and guide future studies on C. semilaevis immunity.

To investigate the roles of microRNAs (miRNA) of Cynoglossus semilaevis in response to Vibrio anguillarum that were previously identified using high-throughput sequencing, microarray analyses was performed on three small RNA libraries (CG, NOSG, and HOSG) prepared from C. semilaevis immune tissues. In total, of 1279 designed probes, 739 (57.78 %) were detectable. The expression levels of these miRNAs were analyzed using pairwise comparisons among the three libraries, and a total of 99 miRNAs were observed to be significantly differentially expressed. The expression patterns of 10 differentially expressed miRNAs were validated by real-time quantitative PCR (RT-qPCR). In addition, expression of miR-142-5p, miR-223, and miR-181a in response to V. anguillarum at numerous time-points in four tissues, as well as the responses to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), peptidoglycan (PGN), and red-spotted grouper nervous necrosis virus (RGNNV) in head kidney cells, were studied by qRT-PCR. Taken together, all of the expression profiles showed significant differences compared to the control group; both similarities and differences in the expression responses to the same pathogen were observed. Collectively, these findings highlighted the putative roles for miRNAs in the context of the innate immune response of C. semilaevis exposing to pathogens and that further studies are needed to understand the molecular mechanisms of miRNA regulation in C. semilaevis host-pathogen interactions.

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most valuable marine aquatic species in Northern China. Given to the rapid development of aquaculture industry, the C. semilaevis was subjected to disease-causing bacteria Vibrio anguillarum. It therefore is indispensable and urgent to understand the mechanism of C. semilaevis host defense against V. anguillarum infection. In the present study, the extensively analysis at the transcriptome level for V. Anguillarum disease in tongue sole was carried out. In total, 94,716 high quality contigs were generated from 75,884,572 clean reads in three libraries (HOSG, NOSG, and CG). 22,746 unigenes were identified when compared with SwissProt, an NR protein database and NT nucleotide database. 954 genes exhibiting the differentially expression at least one pair of comparison in all three libraries were identified. GO enrichment for these genes revealed gene response to biotic stimulus, immune system regulation, and immune response and cytokine production. Further, the pathways such as complement and coagulation cascades and Vibrio cholerae infection pathways were enriched in defensing of pathogen. Besides, 13,428 SSRs and 118,239 SNPs were detected in tongue sole, providing further support for genetic variation and marker-assisted selection in future. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of development of therapeutic regimens and lays the foundation for selecting resistant lines of C. semilaevis against V. anguillarum., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNA) play key regulatory roles in diverse biological processes. Cynoglossus semilaevis is an important commercial mariculture fish species in China. To identify miRNAs and investigate immune-related miRNAs of C. semilaevis, we performed high-throughput sequencing on three small RNA libraries prepared from C. semilaevis immune tissues (liver, head kidney, spleen, and intestine). One library was prepared under normal conditions (control, CG); two were prepared during Vibrio anguillarum infection, where vibriosis symptoms were obvious and non-obvious (HOSG and NOSG, respectively). We obtained 11,216,875, 12,313,404, and 11,398,695 clean reads per library, respectively. Bioinformatic analysis identified 452 miRNAs, including 24 putative novel miRNAs. We analyzed differentially expressed miRNAs between two libraries using pairwise comparison. For NOSG-CG, there was significant differential expression of 175 (38.72%) miRNAs. There was significant differential expression of 215 (47.57%) miRNAs between HOSG and CG. Compared with CG, The HOSG-NOSG comparison revealed significantly different expression of 122 (26.99%) miRNAs respectively. Real-time quantitative PCR (RT-qPCR) experiments were performed for 10 miRNAs of the three samples, and agreement was found between the sequencing and RT-qPCR data. For miRNAs that were significantly differentially expressed, functional annotation of target genes by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that a set of miRNAs that were expressed highly abundantly and significantly differentially were might involved in immune system development and immune response. To our understanding, this is the first report of comprehensive identification of C. semilaevis miRNAs being differentially regulated in immune tissues (liver, head kidney, spleen, and intestine) in normal conditions relating to V. anguillarum infection. Many miRNAs were differentially regulated upon pathogen exposure. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in C. semilaevis host-pathogen interactions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The full length of major histocompatibility complex (MHC) class IIB cDNA was cloned from a Chinese population of Paralichthys olivaceus by homology cloning and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The MHC IIB genomic sequence is 1,864 bp long and consists of 34-bp 5'UTR, 741-bp open reading frame, 407-bp 3'UTR, 96-bp intron1, 392-bp intron2, 85-bp intron3, and 109-bp intron4. Phylogenetic analysis showed that the putative MHC class IIB amino acid of the Chinese P. olivaceus shared 28.3% to 85.4% identity with that of the reported MHC class IIB in other species. A significant association between MHC IIB polymorphism and disease resistance/susceptibility was found in Chinese P. olivaceus. Thirteen different MHC IIB alleles were identified among 411 clones from 84 individuals. Among the 280 (268) nucleotides, 32 (11.4%) nucleotide positions were variable. Most alleles such as alleles a, b, c, d, e, f, j, k, i, m were commonly found in both resistant and susceptible stock. Via chi2 test, allele d was significantly more prevalent in individuals from susceptible stock than from resistant stock, and their percentages were 23.80% and 7.14%, respectively. In addition, allele g occurred in 9 and allele h in 4 of 42 resistant individuals that were not present in the susceptible stock; their percentages were 21.4% and 9.52%, respectively. Although allele l was found only in 8 individuals from the susceptible stock, its percentage is 19.05%.