Characterization and functional analysis of a novel C-type lectin in blunt snout bream (Megalobrama amblycephala).

C-type lectins, one of the pattern recognition receptors (PRRs), play significant roles in innate immune responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. Here, a novel C-type lectin (named as MaCTL) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaCTL is 573 bp long encoding a putative protein of 190 amino acids (aa), which contains a typical feature of signal peptide at 1-23 aa, a characteristic CRD domain at 45-178 aa and a WND/EPN motif that is required for carbohydrates-binding specificity. Phylogenetic analysis indicated that MaCTL is a novel member of CTL family and possessed the highest similarity to that of grass carp (92.11%). The qRT-PCR analysis revealed that MaCTL expressed widely in all examined normal tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the higher expression in the spleen, liver and muscle. The expression of MaCTL in spleen was significantly elevated, peaking at 9 h and 6 h after LPS stimulation and Aeromonas hydrophila challenge, respectively, suggesting its association with involvement in innate immune response. The recombinant MaCTL protein (rMaCTL) agglutinated markedly both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria, including Escherichia coli, Vibrio anguillarum, Vibrio vulnificus and Aeromonas hydrophila, in a Ca2+-dependent manner. Meanwhile, rMaCTL showed the binding effects on the five bacteria and four carbohydrates, such as glucose, surose, LPS and PGN. Moreover, rMaCTL could remarkably inhibit the growth of three types of bacteria in vitro. Overall, the results obtained above demonstrated firmly that MaCTL binds to carbohydrates on the surface of diverse pathogens as a PRR and elicits antimicrobial responses, which shed new light on a better understanding of antibacterial functions of CTLs in teleost fish.

Functions of circular RNAs and their potential applications in gastric cancer (Review).

As the fifth most common cancer and the fourth leading cause of cancer-related death in the world, gastric cancer (GC) poses a potential threat to human health. However, there is still a lack of effective means for the early screening and treatment of GC, and therefore, GC remains a difficult disease to overcome. With the continuous in-depth research on circular RNAs (circRNAs), an increasing body of evidence indicates that circRNAs play an important role in a wide variety of diseases, particularly cancer. Proliferation, invasion and metastatic spread of cancer cells are strongly associated with abnormal circRNA expression. Hence, circRNAs are considered a candidate biomarker for GC diagnosis and prognosis, and a target for cancer treatment. The focus has been on the association of GC with circRNAs, thus it is necessary to briefly review and summarize the relevant research to provide the research findings across the area to researchers, and to indicate the direction for future research. The present review provides an overview on the biogenesis and functions of circRNAs in GC, predicting their possible clinical application as ideal biomarkers and potential targets of treatment in GC.

Toll-interacting protein negatively regulated innate immune response via NF-κB signal pathway in blunt snout bream, Megalobrama amblycephala.

Toll-interacting protein (Tollip) is an important negative regulator of Toll-like receptor-mediated innate immunity by preventing excessive proinflammatory responses. The structure and function of Tollip have been well identified in mammals, but the piscine Tollip remains poorly understood. In the present study, a homologue of Tollip was identified and characterized from blunt snout bream (named MaTollip), which was composed of an 831 bp open reading frame encoding a protein of 276 amino acids. Phylogenetic analysis indicated that MaTollip is a novel member of Tollip family and possessed the highest similarity to that of grass carp (99.28%). Multiple alignment of amino acid sequence showed that MaTOLLIP shared a high degree of structural conservation, including a TBD domain, a C2 domain and a CUE domain, with its counterparts from other vertebrates. With regard to tissue-specific expression without immune challenge, MaTollip was constitutively expressed in a wide range of normal tissues, with the highest in the head-kidney and the lowest in the intestine. MaTollip expression in the head-kidney was strongly upregulated upon LPS stimulation and A. hydrophila infection. Fluorescence microscopic analysis revealed that the green fluorescent protein-TOLLIP was localized predominantly in the cytoplasm of EPC cells in a dot-like state. When MaTollip was overexpressed in HEK-293T and EPC cells, it could significantly inhibit the activity of nuclear factor-κB (NF-κB) promoter in a dose dependent manner. MaTollip overexpression in MAF cells lowered drastically the transcriptional expression level of lipopolysaccharide-induced proinflammatory cytokines (IL-1ß, IL-6 and IL-8), whereas they were dramatically promoted by MaTollip knock down with siRNA. Taken together, this study demonstrated that MaTollip played a pivotal role in mediating host innate immune response to pathogen invasion, and unveiled the involvement of MaTollip in NF-κB-mediated transcription of inflammation genes, which paved the way for further studies of immune negative regulation mechanisms mediated by Tollip in fish.

A novel interleukin-1 receptor-associated kinase 4 from blunt snout bream (Megalobrama amblycephala) is involved in inflammatory response via MyD88-mediated NF-κB signal pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a crucial role in the Toll-like receptor/IL-1R signal pathway, which mediates the downstream signal transduction involved in innate and adaptive immunity. In the present study, an IRAK4 homologue (named as MaIRAK4) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaIRAK4 contains 1422 nucleotides, encoding a putative protein of 473 amino acids. Protein structural analysis revealed that MaIRAK4 has an N-terminal death domain (DD) and a central kinase domain (S_TKc), similar to those of mammals and other fishes. Multiple sequence alignment demonstrated that MaIRAK4 is highly homologous with that of grass carp (97.67%). The qRT-PCR analysis showed that MaIRAK4 expressed widely in all examined tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the highest expression in the liver and spleen. After stimulation with LPS, MaIRAK4 expression upregulated significantly and reached a peak at 6 h and 12 h post LPS stimulation in the spleen and head-kidney, respectively. After challenge with Aeromonas hydrophila, MaIRAK4 expression peaked at 48 h and 72 h in spleen/head-kidney and liver, respectively. These results implied that MaIRAK4 is involved in the host defense against bacterial infection. Subcellular localization analysis indicated that MaIRAK4 distributed in the cytoplasm. Co-immunoprecipitation and subcellular co-localization assay revealed that MaIRAK4 can combine with MaMyD88 through DD domain. MaIRAK4 overexpression can induce slightly the NF-κB promoter activity in HEK 293 cells. However, the activity of NF-κB promoter was dramatically enhanced after co-transfection with MaIRAK4 and MaMyD88 plasmids. The results showed that MaIRAK4 was involved in NF-κB signal pathway mediated by maMyD88. The expression level of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) decreased significantly after the siRNA-mediated knockdown of MaIRAK4. Together, these results suggest that MaIRAK4 plays an important function in the innate immunity of M. amblycephala by inducing cytokines expression.

TAK1 of blunt snout bream promotes NF-κB activation via interaction with TAB1 in response to pathogenic bacteria.

Transforming growth factor-ß activated kinase-1 (TAK1) is an important upstream signaling molecules involved in the NF-κB signaling pathway. TAK1 interacts with TAB1 to form the TAK1-TAB1 complex, which elicits NF-κB activation through a series of cascade reactions in mammals. However, the function of TAK1 in blunt snout bream (Megalobrama amblycephala ( maTak1) and the effects of their interaction between TAK1 and TAB1 on the NF-κB activation still remains largely unknown. In the present study, maTak1 was cloned and characterized successfully based on transcriptome data. Its open reading frame is composed of 1626 nucleotides and the predicted maTAK1 protein contains 541 amino acids, which includes an N-terminal Serine/Threonine protein kinases (S/TKc) and a C-terminal coiled-coil region. Phylogenetic analysis showed that maTAK1 were clustered with those of other teleosts. MaTak1 displayed ubiquitous transcriptional expression in all the examined tissues of healthy blunt snout bream but with varied expression levels. And maTrak1 expression was dramatically enhanced in different tissues and MAF cells after LPS stimulation and A. hydrophila challenge. The result from subcellular localization analysis indicated that both maTAK1 and maTAB1 were cytoplasmic protein. The activity of NF-κB promoter could not be induced by overexpression of maTak1 or maTab1 alone, however, it could be enhanced by co-expression of maTak1 and maTab1. Co-immunoprecipitation and subcellular co-localization assay revealed that maTAK1 can combine with maTAB1 directly. The transcriptional expression level of pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) increased distinctly after the overexpression of maTak1 and maTab1. Taken together, the data obtained in this study demonstrated that the direct interaction between maTAK1 and maTAB1 might play a pivotal role in mediating host innate immune response to pathogen invasion by the production of pro-inflammatory cytokines via NF-κB signaling pathway, which might lay a solid foundation for the establishment of novel therapeutic approach to combat bacterial infection in fish.