外源性绵羊肺腺瘤病毒囊膜蛋白激活MAPK信号通路的分析

doi:doi: 10.11843/j.issn.0366-6964.2016.08.017

Abstract

Abstract:

In order to explore the pathogenesis of exJSRV Env，by the means of the pathology observation and immunohistochemistry，we detected the pathological characteristics and the positive regions of Env，EGFR，p-Erk1/2 and p-p38 in OPA (ovine pulmonary adenomatosis) sheep lung.Then we detected the relative expressions of p-Erk1/2 and p-p38 in sheep lung and in A549 cells after transiently transfecting recombinant plasmid pcDNA4/myc-His/exJSRV-env by Western blot.Finally we detected the proliferation activity of the cells with recombinant plasmid by CCK-8.Immunohistochemistry results revealed that infected lung cells expressed Env in alveolar epithelial cells and tumor cells；EGFR in alveolar epithelial cells，tumor cells slipping off and interstitial cells but less expressed in alveolar epithelial cells and bronchial epithelial cells in normal sheep lung；p-Erk1/2 and p-p38 in alveolar epithelial cells and tumor cells.Western blot results showed that the activation of p-Erk and p-p38 in OPA sheep lung were higher than normal sheep lung.The relative expressions of p-Erk and p-p38 in the A549 cells with recombinant plasmid were higher than control cells.And CCK8 showed that the proliferation activity of the cells with recombinant plasmid were higher than the control cells.We conclude that exJSRV Env activated EGFR/MAPK signal transduction pathway in A549 cells，and possibly promoted the malignant cell proliferation by this pathway and tumorigenesis.We provide a platform for the pathogenesis of JSRV Env.

CLC Number:

S852.659.3

SUN Xiao-lin，DU Fang-yuan，LIU Shu-ying. Recombinant Plasmid pcDNA4/myc-His/exJSRV-env Transiently Transfect A549 Cells and Detect the Activation of MAPK Signal Transduction Pathway[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(8): 1658-1666.

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References

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［2］HOFACRE A，FAN H.Jaagsiekte sheep retrovirus biology and oncogenesis［J］.Viruses，2010，2(12)：2618-2648.
［3］FAN H，PALMARINI M，DEMARTINI J C.Transformation and oncogenesis by jaagsiekte sheep retrovirus［J］.Curr Top Microbiol Immunol，2003，275：139-177.
［4］WOOTTON S K，METZGER M J，HUDKINS K L，et al.Lung cancer induced in mice by the envelope protein of jaagsiekte sheep retrovirus(JSRV)closely resembles lung cancer in sheep infected with JSRV［J］.Retrovirology，2006，3：94.
［5］MCGEE-ESTRADA K，FAN H.In vivo and in vitro analysis of factor binding sites in jaagsiekte sheep retrovirus long terminal repeat enhancer sequences：roles of HNF-3，NF-1，and C/EBP for activity in lung epithelial cells［J］.J Virol，2006，80(1)：332-341.
［6］孔汉金，张克山，刘永杰，等.绵羊肺腺瘤病毒env基因致瘤机制［J］.中国人兽共患病学报，2013，29(5)：504-508.
KONG H J，ZHANG K S，LIU Y J，et al.Oncogenesis mechanism of env gene from Jaagsiekte sheep retrovirus［J］.Chinese Journal of Zoonoses，2013，29(5)：504-508.(in Chinese)
［7］LEROUX C，GIRARD N，COTTIN V，et al.Jaagsiekte sheep retrovirus (JSRV)：from virus to lung cancer in sheep［J］.Vet Res，2007，38(2)：211-228.
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YU M，NI C C，LIU S Q，et al.Expressions of EGFR and pEGFR in non-small cell lung cancer and their clinical significance［J］.Acta Universitatis Medicinalis Anhui，2011，46（1）：57-60.(in Chinese)
［9］蒋璐.PKGⅡ对胃癌细胞由EGF/EGFR启动的信号转导和相关的生物学活动的影响［D］.镇江：江苏大学，2013.
JIANG L.Effect of PKG Ⅱ on EGF/EGFR-induced signal transduction and related biological activities in gastric cancer cells［D］.Zhenjiang：Jiangsu University， 2013.(in Chinese)
［10］徐艳清，廖爱军.p38 MAPK在肿瘤浸润转移中的作用［J］.现代医药卫生，2013，29（20）：3090-3092.
XU Y Q，LIAO A J.The role of p38 lightning in tumor infiltration［J］.Journal of Modern Medicine & Health，2013，29（20）：3090-3092.(in Chinese)
［11］梁佳，包翠芬，魏嘉，等.P38和ERK1/2在肝细胞癌中的表达及意义［J］.中国组织化学与细胞化学杂志，2009，18（2）：202-206.
LIANG J，BAO C F，WEI J，et al.Expression and significance of p38 and ERK1/2 in human heap to cellular carcinoma［J］.Chinese Journal of Histochemistry and Cytochemistry，2009，18（2）：202-206.(in Chinese)
［12］孙佳，王志军，曹平安，等.p38MAPK对肿瘤细胞ATM、ATR和p53信号通路的影响［J］.中华临床医师杂志(电子版)，2013，7（8）：3426-3430.
SUN J，WANG Z J，CAO P A，et al.Effects of p38MAPK towards ATM，ATR and P53 cell signaling patways［J］.Chinese Journal of Clinicians(Electronic Edition)，2013，7（8）：3426-3430.(in Chinese)
［13］MAEDA N，FU W，ORTIN A，et al.Roles of the Ras-MEK–mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-mTOR pathways in Jaagsiekte sheep retrovirus-induced transformation of rodent fibroblast and epithelial cell lines［J］. J Virol，2005，79(7)：4440-4450.
［14］COUSENS C，ALLEAUME C，BIJSMANS E，et al.Jaagsiekte sheep retrovirus infection of lung slice cultures［J］. Retrovirology，2015，12：31.
［15］张亚坤，刘淑英.绵羊肺腺瘤病毒囊膜蛋白致癌作用的研究进展［J］.中国兽医科学，2012，42（12）：1315-1320.
ZHANG Y K，LIU S Y.Advance in tumorigenesis of jaagsiekte sheep retrovirus’ envelope protein［J］.Chinese Veterinary Science，2012，42（12）：1315-1320.(in Chinese)
［16］刘畅，李磊，于立新，等.绵羊肺腺瘤病毒囊膜蛋白及其TM亚基通过激活Akt诱导NIH 3T3细胞转化［J］.中国预防兽医学报，2014，36（8）：606-610.
LIU C，LI L，YU L X，et al.Jaagsiekte retrovirus envelope protein and its transmembrane subunit induced transformation of NIH 3T3 cells by activating Akt［J］.Chinese Journal of Preventive Veterinary Medicine，2014，36（8）：606-610.(in Chinese)
［17］刘畅，敖威华，李磊，等.绵羊肺腺瘤病毒致病机制研究进展［J］.动物医学进展，2015，36（1）：79-82.
LIU C，AO W H，LI L，et al.Progress of tumorigenic mechanisms of ovine pulmonary adenomatosis virus［J］. Progress in Veterinary Medicine，2015，36（1）：79-82.(in Chinese)
［18］LINNERTH-PETRIK N M，SANTRY L A，YU D L，et al.Adeno-associated virus vector mediated expression of an oncogenic retroviral envelope protein induces lung adenocarcinomas in immunocompetent mice［J］.PLoS One，2012，7(12)：e51400.
［19］KETAM S，JOHN F，BRION N，et al.Inhibition of p38 mitogen activated protein kinase increase LPS induced inhibition of apoptosis in neutrophils by activating extracellular signal regulated kinase［J］.Surgery，2001，130(2)：242-247.
［20］JOHNSON C，SANDERS K，FAN H，et al.Jaagsiekte sheep retrovirus transformation in Madin-Darby canine kidney epithelial cell three-dimensional culture［J］. J Virol，2010，84(10)：5379-5390.
［1］PALMARINI M，FAN H.Retrovirus-induced ovine pulmonary adenocarcinoma，an animal model for lung cancer［J］.J Natl Cancer Inst，2001，93(21)：1603-1614.
［2］HOFACRE A，FAN H.Jaagsiekte sheep retrovirus biology and oncogenesis［J］.Viruses，2010，2(12)：2618-2648.
［3］FAN H，PALMARINI M，DEMARTINI J C.Transformation and oncogenesis by jaagsiekte sheep retrovirus［J］.Curr Top Microbiol Immunol，2003，275：139-177.
［4］WOOTTON S K，METZGER M J，HUDKINS K L，et al.Lung cancer induced in mice by the envelope protein of jaagsiekte sheep retrovirus(JSRV)closely resembles lung cancer in sheep infected with JSRV［J］.Retrovirology，2006，3：94.
［5］MCGEE-ESTRADA K，FAN H.In vivo and in vitro analysis of factor binding sites in jaagsiekte sheep retrovirus long terminal repeat enhancer sequences：roles of HNF-3，NF-1，and C/EBP for activity in lung epithelial cells［J］.J Virol，2006，80(1)：332-341.
［6］孔汉金，张克山，刘永杰，等.绵羊肺腺瘤病毒env基因致瘤机制［J］.中国人兽共患病学报，2013，29(5)：504-508.
KONG H J，ZHANG K S，LIU Y J，et al.Oncogenesis mechanism of env gene from Jaagsiekte sheep retrovirus［J］.Chinese Journal of Zoonoses，2013，29(5)：504-508.(in Chinese)
［7］LEROUX C，GIRARD N，COTTIN V，et al.Jaagsiekte sheep retrovirus (JSRV)：from virus to lung cancer in sheep［J］.Vet Res，2007，38(2)：211-228.
［8］于敏，倪琛琛，刘善青，等.EGFR、pEGFR蛋白在非小细胞肺癌中的表达及临床意义［J］.安徽医科大学学报，2011，46（1）：57-60.
YU M，NI C C，LIU S Q，et al.Expressions of EGFR and pEGFR in non-small cell lung cancer and their clinical significance［J］.Acta Universitatis Medicinalis Anhui，2011，46（1）：57-60.(in Chinese)
［9］蒋璐.PKGⅡ对胃癌细胞由EGF/EGFR启动的信号转导和相关的生物学活动的影响［D］.镇江：江苏大学，2013.
JIANG L.Effect of PKG Ⅱ on EGF/EGFR-induced signal transduction and related biological activities in gastric cancer cells［D］.Zhenjiang：Jiangsu University， 2013.(in Chinese)
［10］徐艳清，廖爱军.p38 MAPK在肿瘤浸润转移中的作用［J］.现代医药卫生，2013，29（20）：3090-3092.
XU Y Q，LIAO A J.The role of p38 lightning in tumor infiltration［J］.Journal of Modern Medicine & Health，2013，29（20）：3090-3092.(in Chinese)
［11］梁佳，包翠芬，魏嘉，等.P38和ERK1/2在肝细胞癌中的表达及意义［J］.中国组织化学与细胞化学杂志，2009，18（2）：202-206.
LIANG J，BAO C F，WEI J，et al.Expression and significance of p38 and ERK1/2 in human heap to cellular carcinoma［J］.Chinese Journal of Histochemistry and Cytochemistry，2009，18（2）：202-206.(in Chinese)
［12］孙佳，王志军，曹平安，等.p38MAPK对肿瘤细胞ATM、ATR和p53信号通路的影响［J］.中华临床医师杂志(电子版)，2013，7（8）：3426-3430.
SUN J，WANG Z J，CAO P A，et al.Effects of p38MAPK towards ATM，ATR and P53 cell signaling patways［J］.Chinese Journal of Clinicians(Electronic Edition)，2013，7（8）：3426-3430.(in Chinese)
［13］MAEDA N，FU W，ORTIN A，et al.Roles of the Ras-MEK–mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-mTOR pathways in Jaagsiekte sheep retrovirus-induced transformation of rodent fibroblast and epithelial cell lines［J］. J Virol，2005，79(7)：4440-4450.
［14］COUSENS C，ALLEAUME C，BIJSMANS E，et al.Jaagsiekte sheep retrovirus infection of lung slice cultures［J］. Retrovirology，2015，12：31.
［15］张亚坤，刘淑英.绵羊肺腺瘤病毒囊膜蛋白致癌作用的研究进展［J］.中国兽医科学，2012，42（12）：1315-1320.
ZHANG Y K，LIU S Y.Advance in tumorigenesis of jaagsiekte sheep retrovirus’ envelope protein［J］.Chinese Veterinary Science，2012，42（12）：1315-1320.(in Chinese)
［16］刘畅，李磊，于立新，等.绵羊肺腺瘤病毒囊膜蛋白及其TM亚基通过激活Akt诱导NIH 3T3细胞转化［J］.中国预防兽医学报，2014，36（8）：606-610.
LIU C，LI L，YU L X，et al.Jaagsiekte retrovirus envelope protein and its transmembrane subunit induced transformation of NIH 3T3 cells by activating Akt［J］.Chinese Journal of Preventive Veterinary Medicine，2014，36（8）：606-610.(in Chinese)
［17］刘畅，敖威华，李磊，等.绵羊肺腺瘤病毒致病机制研究进展［J］.动物医学进展，2015，36（1）：79-82.
LIU C，AO W H，LI L，et al.Progress of tumorigenic mechanisms of ovine pulmonary adenomatosis virus［J］. Progress in Veterinary Medicine，2015，36（1）：79-82.(in Chinese)
［18］LINNERTH-PETRIK N M，SANTRY L A，YU D L，et al.Adeno-associated virus vector mediated expression of an oncogenic retroviral envelope protein induces lung adenocarcinomas in immunocompetent mice［J］.PLoS One，2012，7(12)：e51400.
［19］KETAM S，JOHN F，BRION N，et al.Inhibition of p38 mitogen activated protein kinase increase LPS induced inhibition of apoptosis in neutrophils by activating extracellular signal regulated kinase［J］.Surgery，2001，130(2)：242-247.
［20］JOHNSON C，SANDERS K，FAN H，et al.Jaagsiekte sheep retrovirus transformation in Madin-Darby canine kidney epithelial cell three-dimensional culture［J］. J Virol，2010，84(10)：5379-5390.

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Recombinant Plasmid pcDNA4/myc-His/exJSRV-env Transiently Transfect A549 Cells and Detect the Activation of MAPK Signal Transduction Pathway

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