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MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella.

ABSTRACT: MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpressing of cid-miRn-115 and miR-142a-3p results in a visible change in Ctenopharyngodon idella kidney (CIK) cells immune effector activity. Bioinformatics analysis and overexpressing assay shows that cid-miRn-115 and miR-142a-3p directly regulate tlr5 expression. cid-miRn-115 and miR-142a-3p overexpressing leads to a significant decrease in tlr5 expression in CIK, thereby repressing its downstream genes, such as il-1?, il-8 and tnf-?. These findings provide a novel insight into the determination of anti-bacterial compounds in grass carp.

SUBMITTER: Xu XY

PROVIDER: S-EPMC4698583 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella.

Xu Xiao-Yan XY Shen Yu-Bang YB Fu Jian-Jun JJ Yu Hong-Yan HY Huang Wen-Ji WJ Lu Li-Qun LQ Li Jia-Le JL

Scientific reports 20160104

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpre ...[more]

PMID: 26727169

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Project description:BackgroundMyxovirus resistance (Mx) proteins are crucial effectors of the innate antiviral response against a wide range of viruses, mediated by the type I interferon (IFN-I) signaling pathway. However, the antiviral activity of Mx proteins is diverse and complicated in different species.Methodology/principal findingsIn the current study, two novel Mx genes (CiMx1 and CiMx3) were identified in grass carp (Ctenopharyngodon idella). CiMx1 and CiMx3 proteins exhibit high sequence identity (92.1%), and low identity with CiMx2 (49.2% and 49.5%, respectively) from the GenBank database. The predicted three-dimensional (3D) structures are distinct among the three isoforms. mRNA instability motifs also display significant differences in the three genes. The spatial and temporal expression profiles of three C. idella Mx genes and the IFN-I gene were investigated by real-time fluorescence quantitative RT-PCR (qRT-PCR) following infection with grass carp reovirus (GCRV) in vivo and in vitro. The results demonstrated that all the four genes were implicated in the anti-GCRV immune response, that mRNA expression of Mx genes might be independent of IFN-I, and that CIK cells are suitable for antiviral studies. By comparing expression patterns following GCRV challenge or poly(I:C) treatment, it was observed that GCRV blocks mRNA expression of the four genes. To determine the functions of Mx genes, three CiMx cDNAs were cloned into expression vectors and utilized for transfection of CIK cells. The protection conferred by each recombinant CiMx protein against GCRV infection was evaluated. Antiviral activity against GCRV was demonstrated by reduced cytopathic effect, lower virus titer and lower levels of expressed viral transcripts. The transcription of IFN-I gene was also monitored.Conclusions/significanceThe results indicate all three Mx genes can suppress replication of grass carp reovirus and over-expression of Mx genes mediate feedback inhibition of the IFN-I gene.

Project description:Bacillus subtilis is a common group of probiotics that have been widely used in the feed industry as they can increase host resistance to pathogens and balance the immune response. However, the regulatory mechanism of Bacillus subtilis on the host immune system remains unclear in teleosts. In this study, we isolated and enriched dendritic cells from white blood cells (WBCs), and then stimulated them with Bacillus subtilis. Morphological features, specific biological functions, and authorized functional molecular markers were used in the identification of dendritic cells. Subsequently, we collected stimulated cells at 0, 4, and 18 h, and then constructed and sequenced the transcriptomic libraries. A transcriptome analysis showed that 2557 genes were up-regulated and 1708 were down-regulated at 4 h compared with the control group (|Fold Change| ? 4), and 1131 genes were up-regulated and 1769 were down-regulated between the cells collected at 18 h and 4 h (|Fold Change| ? 4). Gene Ontology (GO) annotations suggested many differentially expressed genes (DEGs) (p < 0.05 and |Fold Change| ? 4) were involved in immune-related biological functions including immune system progress, cytokine receptor binding, and cytokine binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the cytokine?cytokine receptor interaction pathways were significantly enriched at both time points (p < 0.05), which may play a key role in the response to stimulation. Furthermore, mRNA expression level examination of several pro-inflammatory cytokines and anti-inflammatory cytokines genes by quantitative real-time polymerase chain reaction (qRT-PCR) indicated that their expressions can be significantly increased in Bacillus subtili, which suggest that Bacillus subtilis can balance immune response and tolerance. This study provides dendritic cell (DC)-specific transcriptome data in grass carp by Bacillus subtilis stimulation, allowing us to illustrate the molecular mechanism of the DC-mediated immune response triggered by probiotics in grass carp.

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MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella.

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MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella.

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