Expression Profile Analysis of miR-221 and miR-222 in Different Tissues and Head Kidney Cells of Cynoglossus semilaevis, Following Pathogen Infection.

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-1ß) 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.

Expression profiling analysis of the microRNA response of Cynoglossus semilaevis to Vibrio anguillarum and other stimuli.

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.

sghC1q, a novel C1q family member from half-smooth tongue sole (Cynoglossus semilaevis): identification, expression and analysis of antibacterial and antiviral activities.

The C1q family includes many proteins that contain a globular (gC1q) domain, and this family is widely conserved from bacteria to mammals. The family is divided into three subgroups: C1q, C1q-like and ghC1q. In this study, a novel C1q family member, sghC1q, was cloned and identified from Cynoglossus semilaevis (named CssghC1q). The full-length CssghC1q cDNA spans 905 bp, including an open reading frame (ORF) of 768 bp, a 5'-untranslated region (UTR) of 25 bp and a 3'-UTR of 112 bp. The ORF encodes a putative protein of 255 amino acids (aa) with a deduced molecular weight of 28 kDa. The predicted protein contains a signal peptide (aa 1-19), a coiled-coil region (aa 61-102) and a globular C1q (gC1q) domain (aa 117-255). Protein sequence alignment indicated that the C-terminus of CssghC1q is highly conserved across several species. Phylogenetic analysis indicated that CssghC1q is most closely related to Maylandia zebra C1q-like-2-like. The CssghC1q genomic sequence spanned 1562 bp, with three exons and two introns. CssghC1q is constitutively expressed in all evaluated tissues, with the highest expression in the liver and the weakest in the heart. After a challenge with Vibrio anguillarum, CssghC1q transcript levels exhibited distinct time-dependent response patterns in the blood, head kidney, skin, spleen, intestine and liver. Recombinant CssghC1q protein exhibited antimicrobial activities against Gram-negative bacteria, Gram-positive bacteria and viruses. The minimum inhibitory concentration (MIC) values against Vibrio harveyi, Vibrio anguillarum, Pseudomonas aeruginosa and Staphylococcus aureus were 0.043 mg/mL, 0.087 mg/mL, 0.174 mg/mL and 0.025 mg/mL, respectively. A low concentration (0.06 mg/mL) of CssghC1q showed significant antiviral activity in vitro against nervous necrosis virus (NNV). These results suggest that CssghC1q plays a vital role in immune defense against bacteria and viruses.

Transcriptome analysis revealed changes of multiple genes involved in immunity in Cynoglossus semilaevis during Vibrio anguillarum infection.

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.

Identification and characterization of Cynoglossus semilaevis microRNA response to Vibrio anguillarum infection through high-throughput sequencing.

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.