chTLR3在传染性法氏囊病病毒感染中的作用及gga-miR-6655-5p对其调控的分子机制
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摘要
为了探讨鸡Toll样受体3(chicken toll-like receptor 3,chTLR3)在传染性法氏囊病病毒(infectious bursal dis-ease virus,IBDV)感染中的作用及microRNA(miRNA)对其调控的分子机制,本研究用IBDV感染DF-1细胞,qRT-PCR和Western blot分析表明IBDV感染可使DF-1细胞中chTLR3的表达水平显著升高。chTLR3激活表达后可抑制IBDV的复制,进一步分析其机理发现,chTLR3表达上调可激活IFN-β启动子和抗病毒天然免疫基因Mx启动子的活性及NF-κB和IRF-7信号通路。用生物信息学方法和双荧光素酶报告系统分析发现gga-miR-6655-5p可作用于chTLR3的3′UTR;qRT-PCR和Western blot分析表明gga-miR-6655-5p可以使chTLR3的蛋白水平表达降低,但mRNA水平没有变化。本研究表明:gga-miR-6655-5p可以作用于chTLR3的3′UTR负调控chTLR3的表达,抑制IFN-β启动子和Mx启动子活性,使IBDV在细胞中的复制水平提高。
The innate immune system acts as the first line of defense against invading viruses and plays an important role in the subsequent activation of antiviral responses.Chicken toll-like receptor 3(chTLR3)is a critical pattern recognition receptors(PRRs)to sense the pathogen-associated molecular patterns(PAMPs)of RNA virus and initial an antiviral innate immune response in chicken.microRNAs(miRNA)are involving in the regulation of chTLR3 to recognize RNA virus infection.However,its role of antiviral activity against infectious bursal disease virus(IBDV)infection and how miRNA regulate chTLR3 in chicken cells both have not been clarified.In this study,we found that the expression of chTLR3 in IBDV-infected-DF-1 cells was significantly increased.Activated chTLR3 signaling via chTLR3 ligand stimulaiton could activate IFN-βpromoter and Mx promoter through IRF7-and NF-κB-dependent pathway and inhibit the replication of IBDV in DF-1 cells,whereas suppression of chTLR3 by siRNA could lead to the contrary results.In addition,we proved that gga-miR-6655-5 p can posttranscriptionally regulate the chTLR3 protein expression level through inhibiting the translation of chTLR3 mRNA based on dual-luciferase reporter system,qRT-PCR and Western blot analysis.Overexpression gga-miR-6655-5 p,reduced the expression of chTLR3 protein,which promoted IBDV replication and decreased the activity of IFN-βpromoter and Mx promoter.In conclusion,Our results suggested that gga-miR-6655-5 p is a negative regulator of chTLR3 and promotes IBDV replication in DF-1 cells.
The innate immune system acts as the first line of defense against invading viruses and plays an important role in the subsequent activation of antiviral responses.Chicken toll-like receptor 3(chTLR3)is a critical pattern recognition receptors(PRRs)to sense the pathogen-associated molecular patterns(PAMPs)of RNA virus and initial an antiviral innate immune response in chicken.microRNAs(miRNA)are involving in the regulation of chTLR3 to recognize RNA virus infection.However,its role of antiviral activity against infectious bursal disease virus(IBDV)infection and how miRNA regulate chTLR3 in chicken cells both have not been clarified.In this study,we found that the expression of chTLR3 in IBDV-infected-DF-1 cells was significantly increased.Activated chTLR3 signaling via chTLR3 ligand stimulaiton could activate IFN-βpromoter and Mx promoter through IRF7-and NF-κB-dependent pathway and inhibit the replication of IBDV in DF-1 cells,whereas suppression of chTLR3 by siRNA could lead to the contrary results.In addition,we proved that gga-miR-6655-5 p can posttranscriptionally regulate the chTLR3 protein expression level through inhibiting the translation of chTLR3 mRNA based on dual-luciferase reporter system,qRT-PCR and Western blot analysis.Overexpression gga-miR-6655-5 p,reduced the expression of chTLR3 protein,which promoted IBDV replication and decreased the activity of IFN-βpromoter and Mx promoter.In conclusion,Our results suggested that gga-miR-6655-5 p is a negative regulator of chTLR3 and promotes IBDV replication in DF-1 cells.
引文
[1]WANG Y S,WANG Z C,TANG Y D,et al.Comparison of four infectious bursal disease viruses isolated from different bird species[J].Arch Virol,2007,152(10):1787-1797.
[2]朱向东,王永山,欧阳伟,等.传染性法氏囊病病毒自然重配株全基因组序列分析[J].中国兽医学报,2014,34(2):219-226.
[3]QIN Y,ZHENG S J.Infectious bursal disease virushost interactions:multifunctional viral proteins that perform multiple and differing jobs[J].Int J Mol Sci,2017,18(1):161.
[4]ALKIE T N,RAUTENSCHLEIN S.Infectious bursal disease virus in poultry:current status and future prospects[J].Vet Med Res Rep,2016(7):9-18.
[5]AKIRA S,UEMATSU S,TAKEUCHI O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[6]THOMPSON A J,LOCARNINI S A.Toll-like receptors,RIG-I-like RNA helicases and the antiviral innate immune response[J].Immunol Cell Biol,2007,85(6):435-445.
[7]KAWAI T,AKIRA S.Antiviral signaling through pattern recognition receptors[J].J Biochem,2007,141(2):137-145.
[8]KARPALA A J,LOWENTHAL J W,BEAN A G.I-dentifying innate immune pathways of the chicken may lead to new antiviral therapies[J].Vet Immunol Immunopathol,2012,148(1/2):100-109.
[9]CHEN S,CHENG A,WANG M.Innate sensing of viruses by pattern recognition receptors in birds[J].Vet Res,2013,44:82.
[10]GUARNIERI D J,DILEONE R J.MicroRNAs:a new class of gene regulators[J].Ann Med,2008,40(3):197-208.
[11]LIM L P,GLASNER M E,YEKTA S,et al.Vertebrate microRNA genes[J].Science,2003,299(5612):1540.
[12]TAKEUCHI O,AKIRA S.Pattern recognition receptors and inflammation[J].Cell,2010,140(6):805-820.
[13]LESTER S N,LI K.Toll-like receptors in antiviral innate immunity[J].J Mol Biol,2014,426(6):1246-1264.
[14]AKIRA S,UEMATSU S,TAKEUCHI O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[15]KAWAI T,AKIRA S.Antiviral signaling through pattern recognition receptors[J].J Biochem,2007,141(2):137-145.
[16]赵霞,张瑞莉,王海彬,等.IBDV感染SPF雏鸡法氏囊中TLR3表达的动态变化[J].黑龙江畜牧兽医,2016,57(5):168-170.
[17]郭新风,王丽琼,李焕荣,等.IBDV感染对SPF鸡腔上囊与外周血单核细胞TLR3/4/15mRNA表达的影响[J].中国兽医杂志,2012,48(10):12-16.
[18]WANG T,TOWN T,ALEXOPOULOU L,et al.Tolllike receptor 3mediates West Nile virus entry into the brain causing lethal encephalitis[J].Nat Med,2004,10(12):1366-1373.
[19]欧阳伟,王永山,王晓丽,等.传染性法氏囊病病毒感染对鸡细胞模式识别受体及抗病毒基因转录的影响[J].中国预防兽医学报,2015,37(2):83-88.
[20]TAURA M,EGUMA A,SUICO M A,et al.p53regulates Toll-like receptor 3expression and function in human epithelial cell lines[J].Mol Cell Biol,2008,28(21):6557-6567.
[21]ONEILL L A,SHEEDY F J,MCCOY C E.MicroR-NAs:the fine-tuners of Toll-like receptor signalling[J].Nat Rev Immunol,2011,11(3):163-75.
[22]NAHID M A,SATOH M,CHAN E K.MicroRNA in TLR signaling and endotoxin tolerance[J].Cell Mol Immunol,2011,8(5):388-403.
[23]SIN O,MABIALA P,LIU Y,et al.Gene Silencing efficiency and INF-βinduction effects of splicing miR-NA 155-Based artificial miRNA with Pre-miRNAstem-loop structures[J].Biochem Genet,2012,50(1/2):112-121.
[24]ZHANG C,HAN L,ZHANG A,et al.Global changes of mRNA expression reveals an increased activity of the interferon-induced signal transducer and activator of transcription(STAT)pathway by repression of miR-221/222in glioblastoma U251cells[J].Int J Oncol,2010,36(6):1503-1512.
[25]QUINN S R,ONEILL L A.A trio of microRNAs that control Toll-like receptor signalling[J].Int Immunol,2011,23(7):421-425.
[26]JIANG C,ZHU W,XU J,et al.MicroRNA-26anegatively regulates toll-like receptor 3expression of rat macrophages and ameliorates pristane induced arthritis in rats[J].Arthritis Res Ther,2014,16(1):R9.
[27]HU X,YE J,QIN A,et al.Both microRNA-155and virus-encoded MiR-155ortholog regulate TLR3expression[J].PLoS One,2015,10(5):e0126012.
[28]LEE C C,WU C C,LIN T L.Chicken melanoma differentiation-associated gene 5(MDA5)recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity[J].Arch Virol,2014,159(7):1671-1686.
[29]LI Y P,HANDBERG K J,JUUL-MADSEN H R,et al.Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus[J].Arch Virol,2007,152(3):463-478.
[30]WONG R T Y,HON C C,ZENG F Y,et al.Screening of differentially expressed transcripts in infectious bursal disease virus-induced apoptotic chicken embryonic fibroblasts by using cDNA microarrays[J].J Gen Virol,2007,88(6):1785-1796.
[31]PEDERSEN I M,CHENG G,WIELAND S,et al.Interferon modulation of cellular microRNA as an antiviral mechanism[J].Nature,2007,449(7164):919-922.
[2]朱向东,王永山,欧阳伟,等.传染性法氏囊病病毒自然重配株全基因组序列分析[J].中国兽医学报,2014,34(2):219-226.
[3]QIN Y,ZHENG S J.Infectious bursal disease virushost interactions:multifunctional viral proteins that perform multiple and differing jobs[J].Int J Mol Sci,2017,18(1):161.
[4]ALKIE T N,RAUTENSCHLEIN S.Infectious bursal disease virus in poultry:current status and future prospects[J].Vet Med Res Rep,2016(7):9-18.
[5]AKIRA S,UEMATSU S,TAKEUCHI O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[6]THOMPSON A J,LOCARNINI S A.Toll-like receptors,RIG-I-like RNA helicases and the antiviral innate immune response[J].Immunol Cell Biol,2007,85(6):435-445.
[7]KAWAI T,AKIRA S.Antiviral signaling through pattern recognition receptors[J].J Biochem,2007,141(2):137-145.
[8]KARPALA A J,LOWENTHAL J W,BEAN A G.I-dentifying innate immune pathways of the chicken may lead to new antiviral therapies[J].Vet Immunol Immunopathol,2012,148(1/2):100-109.
[9]CHEN S,CHENG A,WANG M.Innate sensing of viruses by pattern recognition receptors in birds[J].Vet Res,2013,44:82.
[10]GUARNIERI D J,DILEONE R J.MicroRNAs:a new class of gene regulators[J].Ann Med,2008,40(3):197-208.
[11]LIM L P,GLASNER M E,YEKTA S,et al.Vertebrate microRNA genes[J].Science,2003,299(5612):1540.
[12]TAKEUCHI O,AKIRA S.Pattern recognition receptors and inflammation[J].Cell,2010,140(6):805-820.
[13]LESTER S N,LI K.Toll-like receptors in antiviral innate immunity[J].J Mol Biol,2014,426(6):1246-1264.
[14]AKIRA S,UEMATSU S,TAKEUCHI O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[15]KAWAI T,AKIRA S.Antiviral signaling through pattern recognition receptors[J].J Biochem,2007,141(2):137-145.
[16]赵霞,张瑞莉,王海彬,等.IBDV感染SPF雏鸡法氏囊中TLR3表达的动态变化[J].黑龙江畜牧兽医,2016,57(5):168-170.
[17]郭新风,王丽琼,李焕荣,等.IBDV感染对SPF鸡腔上囊与外周血单核细胞TLR3/4/15mRNA表达的影响[J].中国兽医杂志,2012,48(10):12-16.
[18]WANG T,TOWN T,ALEXOPOULOU L,et al.Tolllike receptor 3mediates West Nile virus entry into the brain causing lethal encephalitis[J].Nat Med,2004,10(12):1366-1373.
[19]欧阳伟,王永山,王晓丽,等.传染性法氏囊病病毒感染对鸡细胞模式识别受体及抗病毒基因转录的影响[J].中国预防兽医学报,2015,37(2):83-88.
[20]TAURA M,EGUMA A,SUICO M A,et al.p53regulates Toll-like receptor 3expression and function in human epithelial cell lines[J].Mol Cell Biol,2008,28(21):6557-6567.
[21]ONEILL L A,SHEEDY F J,MCCOY C E.MicroR-NAs:the fine-tuners of Toll-like receptor signalling[J].Nat Rev Immunol,2011,11(3):163-75.
[22]NAHID M A,SATOH M,CHAN E K.MicroRNA in TLR signaling and endotoxin tolerance[J].Cell Mol Immunol,2011,8(5):388-403.
[23]SIN O,MABIALA P,LIU Y,et al.Gene Silencing efficiency and INF-βinduction effects of splicing miR-NA 155-Based artificial miRNA with Pre-miRNAstem-loop structures[J].Biochem Genet,2012,50(1/2):112-121.
[24]ZHANG C,HAN L,ZHANG A,et al.Global changes of mRNA expression reveals an increased activity of the interferon-induced signal transducer and activator of transcription(STAT)pathway by repression of miR-221/222in glioblastoma U251cells[J].Int J Oncol,2010,36(6):1503-1512.
[25]QUINN S R,ONEILL L A.A trio of microRNAs that control Toll-like receptor signalling[J].Int Immunol,2011,23(7):421-425.
[26]JIANG C,ZHU W,XU J,et al.MicroRNA-26anegatively regulates toll-like receptor 3expression of rat macrophages and ameliorates pristane induced arthritis in rats[J].Arthritis Res Ther,2014,16(1):R9.
[27]HU X,YE J,QIN A,et al.Both microRNA-155and virus-encoded MiR-155ortholog regulate TLR3expression[J].PLoS One,2015,10(5):e0126012.
[28]LEE C C,WU C C,LIN T L.Chicken melanoma differentiation-associated gene 5(MDA5)recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity[J].Arch Virol,2014,159(7):1671-1686.
[29]LI Y P,HANDBERG K J,JUUL-MADSEN H R,et al.Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus[J].Arch Virol,2007,152(3):463-478.
[30]WONG R T Y,HON C C,ZENG F Y,et al.Screening of differentially expressed transcripts in infectious bursal disease virus-induced apoptotic chicken embryonic fibroblasts by using cDNA microarrays[J].J Gen Virol,2007,88(6):1785-1796.
[31]PEDERSEN I M,CHENG G,WIELAND S,et al.Interferon modulation of cellular microRNA as an antiviral mechanism[J].Nature,2007,449(7164):919-922.