猪繁殖与呼吸综合征病毒对Marc145细胞自噬相关基因转录的影响及分析
|
摘要
自噬参与多种疾病过程,且与病毒感染与增殖关系密切。为探究Marc145细胞感染猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)后自噬相关基因Beclin1、ATG5和ATG12mRNA转录差异及规律、PRRSV增殖与细胞自噬的联系;采用实时荧光定量PCR方法对自噬相关基因Beclin1、ATG5和ATG12 mRNA的转录进行测定,绘制了转录规律曲线图;并经实时荧光定量测定并绘制PRRSV在Marc145细胞中增殖规律图。结果显示,试验组自噬相关基因转录量明显高于对照组,表明PRRSV能诱导Marc145细胞自噬,且使Beclin1、ATG5和ATG12mRNA提前进入高转录状态;其转录规律与细胞病变、PRRSV增殖规律呈正相关。
Autophagy is involved in a variety of disease processes and is closely related to viral infection and proliferation.In order to explore the difference and regularity of the mRNA expression of autophagy-related genes Beclin1,ATG5,ATG12 in Marc145 cells infected with porcine reproductive and respiratory syndrome virus(PRRSV),and to correlate PRRSV proliferation with autophagy,the real-time fluorescence quantitative PCR was used.The expressions of Beclin1,ATG5 and ATG12 mRNA were detected by real-time fluorescence quantitative PCR.The proliferation of PRRSV in Marc145 cells was measured by real-time fluorescence.The results showed that the expression of autophagy-related genes in the test group were higher than those in the control group,which means that PRRSV could induce autophagy of Marc145 cells.Meanwhile,the expression of Beclin1,ATG5 and ATG12 mRNA were advanced into the high expression state;the expression principle was positively correlated with the cytopathic effect and PRRSV proliferation.
Autophagy is involved in a variety of disease processes and is closely related to viral infection and proliferation.In order to explore the difference and regularity of the mRNA expression of autophagy-related genes Beclin1,ATG5,ATG12 in Marc145 cells infected with porcine reproductive and respiratory syndrome virus(PRRSV),and to correlate PRRSV proliferation with autophagy,the real-time fluorescence quantitative PCR was used.The expressions of Beclin1,ATG5 and ATG12 mRNA were detected by real-time fluorescence quantitative PCR.The proliferation of PRRSV in Marc145 cells was measured by real-time fluorescence.The results showed that the expression of autophagy-related genes in the test group were higher than those in the control group,which means that PRRSV could induce autophagy of Marc145 cells.Meanwhile,the expression of Beclin1,ATG5 and ATG12 mRNA were advanced into the high expression state;the expression principle was positively correlated with the cytopathic effect and PRRSV proliferation.
引文
[1]LEVINE B,YUAN J Y.Autophagy in cell death:An innocent convict?[J].J Clin Invest,2005,115(10):2679-2788.
[2]CROTZER V L,BLUM J S.Autophagy and intracellular surveillance:Modulating MHC class II antigen presentation with stress[J].Proc Natl Acad Sci U S A,2005,102(22):7779-7780.
[3]SWANLUND J M,KREGEL K C,OBERLEY T D.Investigating autophagy:Quantitative morphometric analysis using electron microscopy[J].Autophagy,2010,6(2):270-277.
[4]KLIONSKY D J,CREGG J M,DUNN W A Jr,et al.A unified nomenclature for yeast autophagy-related genes[J].Dev Cell,2003,5(4):539-545.
[5]KAMADA Y,FUNAKOSHI T,SHINTANI T,et al.Tor-mediated induction of autophagy via an Apg1protein kinase complex[J].J Cell Biol,2000,150(6):1507-1513.
[6]OHSUMI Y.Molecular dissection of autophagy:Two ubiquitin-like systems[J].Nat Rev Mol Cell Biol,2001,2(3):211-216.
[7]WANG K,LI S F,WORKU T,et al.Rab11ais required for porcine reproductive and respiratory syndrome virus induced autophagy to promote viral replication[J].Biochem Biophys Res Commun,2017,492(2):236-242.
[8]LIU Q H,QIN Y X,ZHOU L,et al.Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells[J].Virology,2012,429(2):136-147.
[9]SUN M X,HUANG L,WANG R,et al.Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication[J].Autophagy,2012,8(10):1434-1447.
[10]AITA V M,LIANG X H,MURTY V V V S,et al.Cloning and genomic organization of Beclin1,a candidate tumor suppressor gene on chromosome 17q21[J].Genomics,1999,59(1):59-65.
[11]ZHOU A,LI S F,KHAN A,et al.Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1interaction[J].Virulence,2016,7(2):98-109.
[12]KIMURA S,FUJITA N,NODA T,et al.Monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J].Methods Enzymol,2009,452:1-12.
[13]MIZUSHIMA N,YOSHIMORI T.How to interpret LC3immunoblotting[J].Autophagy,2007,3(6):542-545.
[14]MIJALJICA D,PRESCOTT M,DEVENISH R J.Autophagy in disease[J].Methods Mol Biol,2010,648:79-92.
[15]KURIAKOSE T,KANNEGANTI T D.War on viruses:LC3recruits GTPases[J].Cell Host Microbe,2017,22(1):7-9.
[16]黄剑波.PRRSV SC株GP5蛋白合成肽抗体的制备及ADE作用研究[D].雅安:四川农业大学,2016.HUANG J B.Preparation of GP5protein synthetic peptide antibody of PRRSV SC strain and the research of ADE[D].Yaan:Sichuan Agricultural University,2016.(in Chinese)
[17]MATHEW R,KARANTZA-WADSWORTH V,WHITE E.Role of autophagy in cancer[J].Nat Rev Cancer,2007,7(12):961-967.
[18]WANG T T,CAO Q H,CHEN M Y,et al.Beclin1deficiency correlated with lymph node metastasis,predicts a distinct outcome in intrahepatic and extrahepatic cholangiocarcinoma[J].PLoS One,2013,8(11):e80317.
[19]NASCIMENTO-FERREIRA I,SANTOS-FERREIRA T,SOUSA-FERREIRA L,et al.Overexpression of the autophagic beclin-1protein clears mutant ataxin-3 and alleviates Machado-Joseph disease[J].Brain,2011,134(Pt 5):1400-1415.
[20]KUOS H,TANG G M,MA K,et al.Macroautophagy abnormality in essential tremor[J].PLoS One,2012,7(12):e53040.
[21]PUJHARI S,KRYWORUCHKO M,ZAKHARTCHOUK A N,et al.Role of phosphatidylinositol-3-kinase(PI3K)and the mammalian target of rapamycin(mTOR)signalling pathways in porcine reproductive and respiratory syndrome virus(PRRSV)replication[J].Virus Res,2014,194:138-144.
[22]范许许.自噬复合体ATG5-ATG12促进Ⅰ型干扰素信号转导对口蹄疫病毒复制的影响[D].北京:中国农业科学院,2016.FAN X X.ATG5-ATG12 Conjugate promotes typeⅠIFN signal transduction and influences the replication of foot-and-mouth diease virus[D].Beijing:Chinese Academy of Agricultural Sciences Dissertation,2016.(in Chinese)
[23]LI S F,ZHOU A,WANG J X,et al.Interplay of autophagy and apoptosis during PRRSV infection of Marc145cell[J].Infect Genet Evol,2016,39:51-54.
[24]陈全刚.PRRSV感染诱导细胞自噬及内质网应激的机制研究[D].武汉:华中农业大学,2013.CHEN Q G.The mechanisms study of cellular autophagy and ER stress induced by PRRSV[D].Wuhan:Huazhong Agricultural University,2013.(in Chinese)
[2]CROTZER V L,BLUM J S.Autophagy and intracellular surveillance:Modulating MHC class II antigen presentation with stress[J].Proc Natl Acad Sci U S A,2005,102(22):7779-7780.
[3]SWANLUND J M,KREGEL K C,OBERLEY T D.Investigating autophagy:Quantitative morphometric analysis using electron microscopy[J].Autophagy,2010,6(2):270-277.
[4]KLIONSKY D J,CREGG J M,DUNN W A Jr,et al.A unified nomenclature for yeast autophagy-related genes[J].Dev Cell,2003,5(4):539-545.
[5]KAMADA Y,FUNAKOSHI T,SHINTANI T,et al.Tor-mediated induction of autophagy via an Apg1protein kinase complex[J].J Cell Biol,2000,150(6):1507-1513.
[6]OHSUMI Y.Molecular dissection of autophagy:Two ubiquitin-like systems[J].Nat Rev Mol Cell Biol,2001,2(3):211-216.
[7]WANG K,LI S F,WORKU T,et al.Rab11ais required for porcine reproductive and respiratory syndrome virus induced autophagy to promote viral replication[J].Biochem Biophys Res Commun,2017,492(2):236-242.
[8]LIU Q H,QIN Y X,ZHOU L,et al.Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells[J].Virology,2012,429(2):136-147.
[9]SUN M X,HUANG L,WANG R,et al.Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication[J].Autophagy,2012,8(10):1434-1447.
[10]AITA V M,LIANG X H,MURTY V V V S,et al.Cloning and genomic organization of Beclin1,a candidate tumor suppressor gene on chromosome 17q21[J].Genomics,1999,59(1):59-65.
[11]ZHOU A,LI S F,KHAN A,et al.Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1interaction[J].Virulence,2016,7(2):98-109.
[12]KIMURA S,FUJITA N,NODA T,et al.Monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J].Methods Enzymol,2009,452:1-12.
[13]MIZUSHIMA N,YOSHIMORI T.How to interpret LC3immunoblotting[J].Autophagy,2007,3(6):542-545.
[14]MIJALJICA D,PRESCOTT M,DEVENISH R J.Autophagy in disease[J].Methods Mol Biol,2010,648:79-92.
[15]KURIAKOSE T,KANNEGANTI T D.War on viruses:LC3recruits GTPases[J].Cell Host Microbe,2017,22(1):7-9.
[16]黄剑波.PRRSV SC株GP5蛋白合成肽抗体的制备及ADE作用研究[D].雅安:四川农业大学,2016.HUANG J B.Preparation of GP5protein synthetic peptide antibody of PRRSV SC strain and the research of ADE[D].Yaan:Sichuan Agricultural University,2016.(in Chinese)
[17]MATHEW R,KARANTZA-WADSWORTH V,WHITE E.Role of autophagy in cancer[J].Nat Rev Cancer,2007,7(12):961-967.
[18]WANG T T,CAO Q H,CHEN M Y,et al.Beclin1deficiency correlated with lymph node metastasis,predicts a distinct outcome in intrahepatic and extrahepatic cholangiocarcinoma[J].PLoS One,2013,8(11):e80317.
[19]NASCIMENTO-FERREIRA I,SANTOS-FERREIRA T,SOUSA-FERREIRA L,et al.Overexpression of the autophagic beclin-1protein clears mutant ataxin-3 and alleviates Machado-Joseph disease[J].Brain,2011,134(Pt 5):1400-1415.
[20]KUOS H,TANG G M,MA K,et al.Macroautophagy abnormality in essential tremor[J].PLoS One,2012,7(12):e53040.
[21]PUJHARI S,KRYWORUCHKO M,ZAKHARTCHOUK A N,et al.Role of phosphatidylinositol-3-kinase(PI3K)and the mammalian target of rapamycin(mTOR)signalling pathways in porcine reproductive and respiratory syndrome virus(PRRSV)replication[J].Virus Res,2014,194:138-144.
[22]范许许.自噬复合体ATG5-ATG12促进Ⅰ型干扰素信号转导对口蹄疫病毒复制的影响[D].北京:中国农业科学院,2016.FAN X X.ATG5-ATG12 Conjugate promotes typeⅠIFN signal transduction and influences the replication of foot-and-mouth diease virus[D].Beijing:Chinese Academy of Agricultural Sciences Dissertation,2016.(in Chinese)
[23]LI S F,ZHOU A,WANG J X,et al.Interplay of autophagy and apoptosis during PRRSV infection of Marc145cell[J].Infect Genet Evol,2016,39:51-54.
[24]陈全刚.PRRSV感染诱导细胞自噬及内质网应激的机制研究[D].武汉:华中农业大学,2013.CHEN Q G.The mechanisms study of cellular autophagy and ER stress induced by PRRSV[D].Wuhan:Huazhong Agricultural University,2013.(in Chinese)