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MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

Ashraf U, Zhu B, Ye J, Wan S, Nie Y, Chen Z, Cui M, Wang C, Duan X, Zhang H, Chen H, Cao S - J. Virol. (2016)

Bottom Line: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis.The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation.The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China.

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miR-19b-3p directly targets RNF11. (A) Conservation of the miR-19b-3p target sequence in RNF11 among different species (upper panel), and conservation of the sequence of miR-19b-3p among different species (lower panel). Hsa, Homo sapiens (human); Mmu, Mus musculus (mouse); Rno; Rattus norvegicus (rat). (B) Schematic diagram showing dual-luciferase reporter constructs harboring the 3′ UTR of RNF11 with putative miR-19b-3p binding site. The lower panel shows the alignment of miR-19b-3p and its target site in the 3′ UTR of RNF11. Four mutated nucleotides of the target site are underlined. RL, Renilla luciferase; FL, firefly luciferase. (C) U251 cells were cotransfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM) together with a wild-type or mutated RNF11 3′ UTR dual-luciferase reporter plasmid, and Renilla luciferase activity was measured and normalized to firefly luciferase activity after 24 h. (D and E) U251 cells were transfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM), and then RNF11 mRNA (C) and protein levels (D) were determined after 24 h by quantitative real-time PCR and immunoblotting, respectively. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Protein levels were quantified with immunoblot scanning and normalized to the amount of GAPDH expression.
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Figure 4: miR-19b-3p directly targets RNF11. (A) Conservation of the miR-19b-3p target sequence in RNF11 among different species (upper panel), and conservation of the sequence of miR-19b-3p among different species (lower panel). Hsa, Homo sapiens (human); Mmu, Mus musculus (mouse); Rno; Rattus norvegicus (rat). (B) Schematic diagram showing dual-luciferase reporter constructs harboring the 3′ UTR of RNF11 with putative miR-19b-3p binding site. The lower panel shows the alignment of miR-19b-3p and its target site in the 3′ UTR of RNF11. Four mutated nucleotides of the target site are underlined. RL, Renilla luciferase; FL, firefly luciferase. (C) U251 cells were cotransfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM) together with a wild-type or mutated RNF11 3′ UTR dual-luciferase reporter plasmid, and Renilla luciferase activity was measured and normalized to firefly luciferase activity after 24 h. (D and E) U251 cells were transfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM), and then RNF11 mRNA (C) and protein levels (D) were determined after 24 h by quantitative real-time PCR and immunoblotting, respectively. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Protein levels were quantified with immunoblot scanning and normalized to the amount of GAPDH expression.

Mentions: A previous study has reported RNF11 as a potential target for miR-19b-3p (47). The sequences of miR-19b-3p and its target site in the 3′ UTR of RNF11 were aligned with those from different species, and these sequences are shown to be highly conserved among species (Fig. 4A). These findings were determined using publicly available miRNA target prediction algorithms that include TargetScan, Pictar, and miRanda. To determine if RNF11 mRNA is indeed repressed by miR-19b-3p in the context of virus infection, a dual-luciferase reporter plasmid containing a putative binding site for miR-19b-3p and a mutant construct harboring the miR-19b-3p seed region with a 4-bp mutation were generated (Fig. 4B). A marked reduction in luciferase activity was observed in U251 cells cotransfected with miR-19b-3p mimics and the RNF11 wild-type 3′ UTR carrying a binding site, whereas significantly increased luciferase activity was detected following application of miR-19b-3p inhibitors (Fig. 4C). Moreover, the 4-bp mutation of in the miR-19b-3p seed region led to a complete abrogation of the negative effect of miR-19b-3p on expression of RNF11 3′ UTR reporter constructs (Fig. 4C). These results demonstrate that the nucleotide sequence in the 3′ UTR of RNF11 is a potential miR-19b-3p targeting site. To further substantiate that RNF11 is indeed a target of miR-19b-3p, endogenous RNF11 expression was determined in U251 cells treated with miR-19b-3p mimics or inhibitors. As shown in Fig. 4D and E, the ectopic expression of miR-19b-3p significantly repressed RNF11 mRNA and protein levels, whereas miR-19b-3p inhibitors restored RNF11 expression, indicating that RNF11 expression could be squelched by miR-19b-3p via mRNA decay and translational suppression. Thus, these data suggest that RNF11 is a direct target of miR-19b-3p and that its expression is modulated by miR-19b-3p.


MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

Ashraf U, Zhu B, Ye J, Wan S, Nie Y, Chen Z, Cui M, Wang C, Duan X, Zhang H, Chen H, Cao S - J. Virol. (2016)

miR-19b-3p directly targets RNF11. (A) Conservation of the miR-19b-3p target sequence in RNF11 among different species (upper panel), and conservation of the sequence of miR-19b-3p among different species (lower panel). Hsa, Homo sapiens (human); Mmu, Mus musculus (mouse); Rno; Rattus norvegicus (rat). (B) Schematic diagram showing dual-luciferase reporter constructs harboring the 3′ UTR of RNF11 with putative miR-19b-3p binding site. The lower panel shows the alignment of miR-19b-3p and its target site in the 3′ UTR of RNF11. Four mutated nucleotides of the target site are underlined. RL, Renilla luciferase; FL, firefly luciferase. (C) U251 cells were cotransfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM) together with a wild-type or mutated RNF11 3′ UTR dual-luciferase reporter plasmid, and Renilla luciferase activity was measured and normalized to firefly luciferase activity after 24 h. (D and E) U251 cells were transfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM), and then RNF11 mRNA (C) and protein levels (D) were determined after 24 h by quantitative real-time PCR and immunoblotting, respectively. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Protein levels were quantified with immunoblot scanning and normalized to the amount of GAPDH expression.
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Figure 4: miR-19b-3p directly targets RNF11. (A) Conservation of the miR-19b-3p target sequence in RNF11 among different species (upper panel), and conservation of the sequence of miR-19b-3p among different species (lower panel). Hsa, Homo sapiens (human); Mmu, Mus musculus (mouse); Rno; Rattus norvegicus (rat). (B) Schematic diagram showing dual-luciferase reporter constructs harboring the 3′ UTR of RNF11 with putative miR-19b-3p binding site. The lower panel shows the alignment of miR-19b-3p and its target site in the 3′ UTR of RNF11. Four mutated nucleotides of the target site are underlined. RL, Renilla luciferase; FL, firefly luciferase. (C) U251 cells were cotransfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM) together with a wild-type or mutated RNF11 3′ UTR dual-luciferase reporter plasmid, and Renilla luciferase activity was measured and normalized to firefly luciferase activity after 24 h. (D and E) U251 cells were transfected with miR-19b-3p mimics, miR-19b-3p inhibitors, or the corresponding control oligonucleotide (final concentration, 50 nM), and then RNF11 mRNA (C) and protein levels (D) were determined after 24 h by quantitative real-time PCR and immunoblotting, respectively. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Protein levels were quantified with immunoblot scanning and normalized to the amount of GAPDH expression.
Mentions: A previous study has reported RNF11 as a potential target for miR-19b-3p (47). The sequences of miR-19b-3p and its target site in the 3′ UTR of RNF11 were aligned with those from different species, and these sequences are shown to be highly conserved among species (Fig. 4A). These findings were determined using publicly available miRNA target prediction algorithms that include TargetScan, Pictar, and miRanda. To determine if RNF11 mRNA is indeed repressed by miR-19b-3p in the context of virus infection, a dual-luciferase reporter plasmid containing a putative binding site for miR-19b-3p and a mutant construct harboring the miR-19b-3p seed region with a 4-bp mutation were generated (Fig. 4B). A marked reduction in luciferase activity was observed in U251 cells cotransfected with miR-19b-3p mimics and the RNF11 wild-type 3′ UTR carrying a binding site, whereas significantly increased luciferase activity was detected following application of miR-19b-3p inhibitors (Fig. 4C). Moreover, the 4-bp mutation of in the miR-19b-3p seed region led to a complete abrogation of the negative effect of miR-19b-3p on expression of RNF11 3′ UTR reporter constructs (Fig. 4C). These results demonstrate that the nucleotide sequence in the 3′ UTR of RNF11 is a potential miR-19b-3p targeting site. To further substantiate that RNF11 is indeed a target of miR-19b-3p, endogenous RNF11 expression was determined in U251 cells treated with miR-19b-3p mimics or inhibitors. As shown in Fig. 4D and E, the ectopic expression of miR-19b-3p significantly repressed RNF11 mRNA and protein levels, whereas miR-19b-3p inhibitors restored RNF11 expression, indicating that RNF11 expression could be squelched by miR-19b-3p via mRNA decay and translational suppression. Thus, these data suggest that RNF11 is a direct target of miR-19b-3p and that its expression is modulated by miR-19b-3p.

Bottom Line: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis.The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation.The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China.

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Related in: MedlinePlus