miR-2187 mimics induces a significant decrease in luciferase activity only in wild-type TRAF6 3UTR (Figure 3C)

miR-2187 mimics induces a significant decrease in luciferase activity only in wild-type TRAF6 3UTR (Figure 3C). and rhabdoviru(SCRV) can up-regulate the expression MADH3 of miR-2187. Elevated miR-2187 is capable of reducing the production of inflammatory factors and antiviral genes by targeting TRAF6, thereby avoiding excessive inflammatory response. Furthermore, we proved that miR-2187 modulates innate immunity through TRAF6-mediated NF-B and IRF3 signaling pathways. The above results indicate that miR-2187 acts as an immune inhibitor involved in host antibacterial and antiviral responses, thus enriching the immune regulatory network of the conversation between host and pathogen in lower vertebrates. is usually a Gram-negative pathogen, while SCRV is usually a member of rhabdovirus, a kind of fish RNA computer virus. Both of these pathogens can cause severe hemorrhagic septicemia according to reports (29). Therefore, the regulation mechanism of and SCRV contamination in teleost is the Mps1-IN-3 focus of our research. In the present study, we statement a new regulatory mechanism of miRNA in response to innate immunity. We have explored the expression of miR-2187 and the relationship between miR-2187 and TRAF6 under the stimulation of Gram-negative bacteria or rhabdovirus (SCRV), a typical fish RNA rhabdovirus. Importantly, we found that miiuy croaker miR-2187 could be rapidly upregulated after (1.5 108 CFU/ml), LPS (InvivoGen, 1mg/ml), poly(I:C) (InvivoGen, 1mg/ml), or SCRV at a multiplicity of infection (MOI) of 5 through intraperitoneal route, and individual challenged with 100 l of physiological saline as a comparison group. After that, the fish were killed at different time points and the spleen tissues were collected for RNA extraction. All animal experimental procedures were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Research Ethics Committee of Shanghai Ocean University. Cell Culture and Transfection Epithelioma papulosum cyprinid (EPC) cells were cultured in medium 199 (Invitrogen) supplemented containing 10% FBS, 1% Penicillin-Streptomycin Solution (100) under condition with 5% CO2 at 26C. Cells with no stimulation were collected as the control, and each experiment have three biological replicates. Miiuy croaker macrophages were aseptically isolated from the head kidney samples as described (30). The cells were cultured in L-15 (Hyclone) medium supplemented with 15% FBS (Life Technologies) and 1% Penicillin-Streptomycin Solution (100). Miiuy croaker kidney cell lines Mps1-IN-3 (MKC) were cultured in incubator at 26C. Cells were divided into 24-well or 48-well plates before they were transferred until 80% of cell density. Prior to transient transfection, cells were seeded into each well of a 24-well or 48-well plate and incubated overnight. Subsequently, EPC cells were transfected with the plasmid using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s protocol. RNA oligoribonucleotides were transfected into MKC cells by using Lipofectamine RNAiMAX (Invitrogen). Washing the macrophages and infecting them with LPS, poly(I:C) or SCRV with MOI of 5, and incubate at different times as indicated. Plasmid Construction In order to construct the TRAF6 expression plasmid, the full-length coding sequence (CDS) region and 3-untranslated regions (3UTR) Mps1-IN-3 of the miiuy croaker TRAF6 gene were amplified by specific primer pairs and restricted endonuclease sites III and I, and then inserted into pcDNA3.1 vector (Invitrogen) with a Flag tag. To construct a TRAF6 3-UTR plasmid, the full-length TRAF6 3-UTR region of were cloned into pmir-GLO luciferase reporter vector to construct the wild type TRAF6-3UTR plasmid. The mutant-types of the TRAF6 3-UTR reporter vector were Mps1-IN-3 conducted by using Mut Express II Fast Mutagenesis Kit V2 (Vazyme) with mutant primers. Additionally, the wild type of miiuy croaker TRAF6 3-UTR or the mutant-type was cloned into the mVenus-C1 vector (Invitrogen) which contained.