Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: Identification of MIR122HG. A , expression patterns of MIR122HG at the indicated time after SCRV infection in miiuy croaker spleen samples measured by qRT–PCR. B , expression patterns of MIR122HG at the indicated time after SCRV infection in MICs measured by qRT–PCR. C , relative expression of MIR122HG in indicated cell lines was determined by qRT–PCR, and the expression of MIR122HG in MIC cell line was used as control. D , schematic of the MIR122HG locus. MIR122HG locates on miiuy croaker chromosome 18. E , MIR122HG was predicted to be ncRNAs. The RNA sequences of MIR122HG were put into the coding potential calculator (CPC) program, which was predicted to be ncRNAs. F , MIR122HG was verified to be ncRNA by Western blotting. TAK1-FLAG plasmid and MIR122HG-FLAG plasmid were transfected into MICs, respectively, and the expression was detected by Western blotting. All data presented as the means ± SD from at least three independent triplicated experiments. ∗∗ p < 0.01; ∗ p < 0.05. hsa-TAK1, Homo sapiens TAK1 gene; MIC, Miichthys miiuy intestine cell; mmi-TAK1, Miichthys miiuy TAK1 gene; ncRNA, noncoding RNA; qRT–PCR, quantitative RT–PCR; SCRV, Siniperca chuatsi rhabdovirus.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Western Blot, Plasmid Preparation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: MIR122HG suppresses host antiviral immunity. A , schematic diagram of MIR122HG expression plasmid construction. B , location schematic diagram of si-MIR122HG and efficient si-MIR122HG was screened. The three siRNAs of MIR122HG was transfected into MSpCs for 48 h, respectively. C , effect of the expression plasmid of MIR122HG on TNF-α, IL-8, IFN-2, and Mx1 expression during SCRV infection. MICs were transfected with pcDNA3.1 vector or MIR122HG expression plasmid for 24 h and then treated with SCRV for 6, 12, or 24 h; subsequently, expression of the indicated genes was determined by qPCR. D , MSpCs were transfected with si-NC or si-MIR122HG for 24 h. The cells were treated with SCRV for different times. Then, the expression of TNF-α, IL-8, IFN-2, and Mx1 were analyzed by qPCR. E and F , cell proliferation was assessed by EdU assays in MICs transfected with pcDNA3.1 vector or MIR122HG expression plasmid ( E ) and si-NC or si-MIR122HG ( F ) to determine the effect of MIR122HG after SCRV infection. MICs were transfected with si-NC, si-MIR122HG, pcDNA3.1 vector, or MIR122HG expression plasmid for 24 h and then treated with SCRV for 24 h. A cell proliferation assay was performed. The scale bar represents 20 μm. All data presented as the means ± SD from at least three independent triplicated experiments. ∗∗ p < 0.01; ∗ p < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; IFN-2, interferon 2; IL-8, interleukin 8; MIC, Miichthys miiuy intestine cell; MSpC, Miichthys miiuy spleen cell; NC, negative control; qPCR, quantitative PCR; SCRV, Siniperca chuatsi rhabdovirus; TNF-α, tumor necrosis factor alpha.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Expressing, Plasmid Preparation, Transfection, Infection, Proliferation Assay, Negative Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: MIR122HG regulates the expression of miR-122-5p. A and B , expression patterns of miR-122-5p at the indicated time after SCRV infection in miiuy croaker spleen samples ( A ) and MSpCs ( B ) measured by qRT–PCR. C , MICs were transfected with pcDNA3.1, MIR122HG, MIR122HG-MT, NC, or si-MIR122HG for 48 h, and then the expression profiles of MIR122HG and miR-122-5p were detected by qRT–PCR, respectively. All data were presented as the means ± SD from at least three independent triplicated experiments. ∗∗ p < 0.01; ∗ p < 0.05. MIC, Miichthys miiuy intestine cell; MSpC, Miichthys miiuy spleen cell; NC, negative control; qRT–PCR, quantitative RT–PCR; SCRV, Siniperca chuatsi rhabdovirus.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Expressing, Infection, Quantitative RT-PCR, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: miR-122-5p targets miiuy croaker TAK1 gene. A , schematic diagram of the predicted target sites of miR-122-5p in the 3′-UTR of TAK1. miR-122-5p-binding sites in the WT of TAK1 and the mutant type were shown. B , miR-122-5p regulates TAK1 expression. EPC cells were cotransfected with the TAK1 expression plasmid, along with miR-122-5p or NC, pcDNA3.1 or MIR122HG. After 48 h, TAK1 expression was determined by Western blotting ( lower panel ), and the gray value ratio about TAK1/tubulin is shown in the upper panel . C , MICs were cotransfected with the concentration gradient of NC (100, 75, 50, and 0 nM) or miR-122-5p (0, 25, 50, and 100 nM), pcDNA3.1 (200, 150, 100, and 0 ng) or MIR122HG (0, 50, 100, and 200 ng) for 24 h. Then the levels of TAK1 were measured by qRT–PCR. D , EPC cells were transfected with the WT or the mutant type of TAK1 3′-UTR, along with NC or miR-122-5p for 24 h, and then the luciferase activity was measured, respectively. E and F , EPC cells were cotransfected with the WT or mutant type of mVenus-TAK1-3′UTR with the NC or miR-122-5p, respectively. After 48 h of transfection, the fluorescence intensity was evaluated by an enzyme-labeled instrument ( E ), and the GFP expression ( F ) was measured by Western blotting, respectively. The scale bar represents 20 μm; original magnification ×10. G , EPC cells were transfected with the WT or the mutant type of TAK1 3′-UTR, along with pcDNA3.1 or MIR122HG for 24 h, and then the luciferase activity was measured, respectively. H and I , EPC cells were cotransfected with the WT or mutant type of mVenus-TAK1-3′UTR with the pcDNA3.1 or MIR122HG, respectively. After 48 h of transfection, the fluorescence intensity was evaluated by an enzyme-labeled instrument ( H ), and the GFP expression ( I ) was measured by Western blotting, respectively. The scale bar represents 20 μm; original magnification ×10. All data were presented as the means ± SD from at least three independent triplicated experiments. ∗∗ p < 0.01; ∗ p < 0.05. EPC, epithelioma papulosum cyprini; MIC, Miichthys miiuy intestine cell; NC, negative control; qRT–PCR, quantitative RT–PCR; TAK1, transforming growth factor-β–activated kinase 1.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Binding Assay, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Concentration Assay, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Fluorescence, Labeling, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: MIR122HG negatively regulates TAK1-mediated antiviral signaling. A , MIR122HG inhibits the mRNA level of endogenous TAK1 upon SCRV infection. qPCR assays were performed to determine the expression levels of TAK1 in MICs transfected with pcDNA3.1, MIR122HG, NC, or si-MIR122HG at different SCRV infection times. B , MIR122HG suppresses the protein expression of endogenous TAK1 upon SCRV infection. MICs were transfected with pcDNA3.1 or MIR122HG at different concentrations, and MSpCs were transfected with NC or si-MIR122HG for 24 h. Then the cells were treated with SCRV for 24 h, and the expression of TAK1 was determined by Western blotting ( lower panel ), and the gray value ratio about TAK1/tubulin is shown in the upper panel . C , MIR122HG inhibits TAK1-activated luciferase activity. EPC cells were cotransfected with pcDNA3.1, MIR122HG, or MIR122HG-MT, a TAK1 expression plasmid, and a pRL-TK vector, along with an NF-κB, IL-8, IFN-2, or IRF3 reporter gene, to investigate the regulatory effect of MIR122HG on NF-κB and IRF3 signals. D , relative luciferase activity of the indicated reporters in EPC cells after cotransfection with pcDNA3.1 (200, 150, 100, and 0 ng) or MIR122HG (0, 50, 100, and 200 ng) and TAK1 expression plasmid, pRL-TK vector, together with NF-κB, IL-8, IFN-2, or IRF3 luciferase reporter genes, and then the luciferase activity was measured after 24 h. E , relative luciferase activity of the indicated reporters in EPC cells after cotransfection with pcDNA3.1 or MIR122HG was determined. EPC cells were cotransfected with pcDNA3.1 or MIR122HG plasmid, TAK1 expression plasmid, pRL-TK vector, together with NF-κB, IL-8, IFN-2, or IRF3 luciferase reporter genes, and then the luciferase activity was measured at different time points, as indicated. All data were presented as the means ± SD from at least three independent triplicated experiments. ∗∗ p < 0.01; ∗ p < 0.05. EPC, epithelioma papulosum cyprini; IFN-2, type 2 interferon; IL-8, interleukin 8; IRF3, interferon regulatory factor 3; MIC, Miichthys miiuy intestine cell; MSpC, Miichthys miiuy spleen cell; MT, mutant type; NC, negative control; qPCR, quantitative PCR; SCRV, Siniperca chuatsi rhabdovirus; TAK1, transforming growth factor-β–activated kinase 1.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Infection, Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Cotransfection, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: LncRNA MIR122HG promotes SCRV replication. A , MIR122HG promotes SCRV replication. MSpCs transfected with NC or si-MIR122HG and with pcDNA3.1 or MIR122HG or MIR122HG-MT for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. B , MSpCs were transfected with NC or si-MIR122HG, and MICs were transfected with pcDNA3.1 or MIR122HG for 24 h and infected with SCRV at MOI 5 for 1 h and washed and then added with fresh medium. After 48 and 72 h, SCRV TCID 50 in cultural supernatants was measured with MSpC and MICs. C , miR-122-5p promotes SCRV replication. MSpCs transfected with NC or miR-122-5p and with NC-i or miR-122-5p-i for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. D , MICs were transfected with NC or miR-122-5p, and MSpCs were transfected with NC-i or miR-122-5p-i for 24 h and infected with SCRV at MOI 5 for 1 h and washed and then added with fresh medium. After 48 and 72 h, SCRV TCID 50 in cultural supernatants was measured with MSpCs and MICs. All data are presented as the means ± SD from at least three independent triplicated experiments. ∗∗ p < 0.01; ∗ p < 0.05. LncRNA, long noncoding RNA; MIC, Miichthys miiuy intestine cell; miR-122-5p-i, miR-122-5p inhibitor; MOI, multiplicity of infection; MSpC, Miichthys miiuy spleen cell; MT, mutant type; NC, negative control; NC-i, NC inhibitor; qPCR, quantitative PCR; SCRV, Siniperca chuatsi rhabdovirus; TCID 50 , 50% tissue culture infectious dose.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Transfection, Infection, RNA Expression, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish

doi: 10.1016/j.jbc.2022.101773

Figure Lengend Snippet: MIR122HG regulating TAK1 gene is widely found in fish. A , sequence alignment of MIR122HG from various species where it is present; miR-122-5p sequences are shown in the black box . B and C , schematic diagram of the predicted target sites of miR-122-5p in the 3′ UTRs of Larimichthys crocea TAK1 ( B ) ( left ) and Nibea diacanthus TAK1 ( C ) ( left ). EPC cells were transfected with pcDNA3.1 (200, 150, 100, and 0 ng) or Lcr MIR122HG (0, 50, 100, and 200 ng) along with the WT L. crocea TAK1 3′ UTR ( Lcr TAK1 3′-UTR-WT) ( B ) ( middle ). EPC cells were transfected with pcDNA3.1 or Lcr MIR122HG along with the WT L. crocea TAK1 3′ UTR ( Lcr TAK1 3′-UTR-WT) or the mutant ( Lcr TAK1 3′-UTR-MT) for 24 h, and then the luciferase activity was determined ( B ) ( right ). EPC cells were transfected with pcDNA3.1 (200, 150, 100, and 0 ng) or Ndi MIR122HG (0, 50, 100, and 200 ng) along with the WT N. diacanthus TAK1 3′ UTR ( Ndi TAK1 3′-UTR-WT) ( C ) ( middle ). EPC cells were transfected with pcDNA3.1 or Ndi MIR122HG along with the WT N. diacanthus TAK1 3′ UTR ( Ndi TAK1 3′-UTR-WT) or the mutant ( Ndi TAK1 3′-UTR-MT) for 24 h, and then the luciferase activity was determined ( C ) ( right ). D , mechanism diagram of TAK1 regulatory network and their function. MIR122HG could regulate the release of miR-122-5p and act as the precursor of miR-122-5p to indirect suppress the expression of TAK1, thus may promote the escape of bacteria. All data were presented as the means ± SD from at least three independent triplicate experiments. ∗∗ p < 0.01; ∗ p < 0.05. EPC, epithelioma papulosum cyprini; MT, mutant type; TAK1, transforming growth factor-β–activated kinase 1.

Article Snippet: Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).

Techniques: Sequencing, Transfection, Mutagenesis, Luciferase, Activity Assay, Expressing, Bacteria