弓形虫H4和Rab2基因真核表达载体的构建及其对N2a细胞凋亡和自噬的影响
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摘要
本研究旨在探索弓形虫Rab2和H 4基因在神经细胞凋亡和自噬过程中的作用,并进一步研究雷帕霉素(rapamycin,RAPA)对其的影响。本试验以弓形虫M e49虫株c DN A为模板,扩增H4和Rab2基因,并构建到真核表达载体pcDNA3.1 (+)上,转染小鼠神经母细胞瘤细胞(N2a),同时设置RAPA给药组,检测目的基因及凋亡和自噬相关蛋白的表达情况。结果显示,重组质粒成功构建,弓形虫Rab2和H4基因在N2a细胞中表达,导致细胞凋亡相关蛋白Bcl-2下调和细胞自噬相关蛋白LC3Ⅰ/Ⅱ上调,但在RAPA组,Bcl-2却有上调趋势。此外还发现H4基因对于Rab2表达有促进作用。结果表明,H4和Rab2的表达可以促进N2a细胞的凋亡和自噬,而RAPA具有抑制细胞凋亡的作用,从而保护神经系统,这为弓形虫病的预防治疗提供了新思路。
Toxoplasmosis is a fatal disease mediated by Toxoplasma gondii invasion in the central nervous system(CNS). We aimed to find out the role of Rab2 and H4 genes of T.gondii in the process of neuronal apoptosis and autophagy, and the effect of rapamycin(RAPA). By using the c DNA of T.gondii Me49 strain as a template, H4 and Rab2 genes were amplified and were linked to the eukaryotic expression vector pc DNA3.1(+), then were transfected into mouse neuroblastoma N2 a cells(N2 a) by lipofectamine2000.We set up a RAPA group and detected the expression of the target genes, the apoptotic and autophagic proteins. In this study, the recombinant plasmids of pc DNA3.1(+)-HA-H4 and pc DNA3.1(+)-HA-Rab2 were constructed successfully, and were expressed in N2 a cells, where the expression levels of both genes were significantly higher than the control group(P<0.01).In the transfected group, the expression of Bcl-2 gene had been on a downward trend, and the expression of LC3 gene had been on an upward trend, but the expression of Bcl-2 increased after adding with PAPA. Moreover, co-transfection of pc DNA3.1(+)-HA-Rab2 and pcDNA3.1(+)-HA-H4 showed that H4 gene could promote Rab2 expression.Specifically, we found that H4 and Rab2 proteins can promote the apoptosis and autophagy of N2a cells,which can be inhibited the apoptosis by the RAPA. Because of the CNS protection by adding PAPA,this study might provide a new idea for the prevention and treatment of animal toxoplasmosis.
Toxoplasmosis is a fatal disease mediated by Toxoplasma gondii invasion in the central nervous system(CNS). We aimed to find out the role of Rab2 and H4 genes of T.gondii in the process of neuronal apoptosis and autophagy, and the effect of rapamycin(RAPA). By using the c DNA of T.gondii Me49 strain as a template, H4 and Rab2 genes were amplified and were linked to the eukaryotic expression vector pc DNA3.1(+), then were transfected into mouse neuroblastoma N2 a cells(N2 a) by lipofectamine2000.We set up a RAPA group and detected the expression of the target genes, the apoptotic and autophagic proteins. In this study, the recombinant plasmids of pc DNA3.1(+)-HA-H4 and pc DNA3.1(+)-HA-Rab2 were constructed successfully, and were expressed in N2 a cells, where the expression levels of both genes were significantly higher than the control group(P<0.01).In the transfected group, the expression of Bcl-2 gene had been on a downward trend, and the expression of LC3 gene had been on an upward trend, but the expression of Bcl-2 increased after adding with PAPA. Moreover, co-transfection of pc DNA3.1(+)-HA-Rab2 and pcDNA3.1(+)-HA-H4 showed that H4 gene could promote Rab2 expression.Specifically, we found that H4 and Rab2 proteins can promote the apoptosis and autophagy of N2a cells,which can be inhibited the apoptosis by the RAPA. Because of the CNS protection by adding PAPA,this study might provide a new idea for the prevention and treatment of animal toxoplasmosis.
引文
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[14]STENMARK H,OLKKONEN V M.The Rab GTPase family[J].Genome Biology,2001,2(5):S3007.
[15]MOHRMANN K,VAN DER SLUIJS P.Regulation of membrane transport through the endocytic pathway by rab GTPases[J].Molecular Membrane Biology,1999,16(1):81-87.
[16]STENMARK H.Rab GTPases as coordinators of vesicle traffic[J].Nature Reviews Molecular Cell Biology,2009,10(8):513-525.
[17]KHOKHA R,MURTHY A,WEISS A.Metalloproteinases and their natural inhibitors in inflammation and immunity[J].Nature Reviews Immunology,2013,13(9):649-665.
[18]陈华萍,陈旭昕,董伟杰,等.Rab26诱导宫颈癌HeLa细胞凋亡并抑制其迁移[J].第三军医大学学报,2016(24):2576-2579.CHEN Hua-ping,CHEN Xu-xin,DONG Wei-jie,et al.Rab26 induces apoptosis and suppresses migration of He La cells[J].Journal of Third Military Medical University,2016(24):2576-2579.(in Chinese)
[19]李小山,李国利,冯晓灵,等.过表达Rab27B体外促进人胃癌细胞系MKN-45凋亡并抑制其增殖[J].基础医学与临床,2016(7):976-980.LI Xiao-shan,LI Guo-li,FENG Xiao-ling,et al.Overexpressed Rab27B induces apoptosis and inhibits proliferation of MKN45 cell line in vitro[J].Basic&Clinical Medicine,2016(7):976-980.(in Chinese)
[20]ROHDE J R,CARDENAS M E.The tor pathway regulates gene expression by linking nutrient sensing to histone acetylation[J].Molecular and Cel lular Biology,2003,23(2):629-635.
[21]YU Z,KONE B C.Targeted histone H4 acetylation via phosphoinositide 3-kinase-and p70s6-kinase-dependent pathways inhibits iNOS induction in mesangial cells[J].American Journal of Physiolo gyRenal Physiology,2006,290(2):496-502.
[22]DURAN R V,HALL M N.Regulation of TOR by small GT-Pases[J].EMBO Reports,2012,13(2):121-128.
[23]LI L,KIM E,YUAN H,et al.Regulation of mTORC1 by the Rab and Arf GTPases[J].Journal of Biological Chemistry,2010,285(26):19705-19709.
[24]MATSUI T,FUKUDA M.Rab12.regulates mTORC1 activity and autophagy through controlling the degradation of amino-acid transporter PAT4[J].EMBOReports,2013,14(5):450-457.
[25]CARGNELLO M,TCHERKEZIAN J,ROUX P P.The expanding role of mTOR in cancer cell growth and proliferation[J].Mutagenesis,2015,30(2):169-176.
[26]CACCAMO A,MAJUMDER S,RICHARDSON A,et al.Molecular interplay between mammalian target of rapamycin(mTOR),amyloid-β,and Tau[J].Journal of Biological Chemistry,2010,285(17):13107-13120.
[27]CACCAMO A,MALDONADO M A,MAJUMDER S,et al.Naturally secreted amyloid-βincreases mammalian target of rapamycin(mTOR)activity via a PRAS40-mediated mechanism[J].Journal of Biological Chemistry,2011,286(11):8924-8932.
[28]CASADIO A,MARTIN K C,GIUSTETTO M,et al.A transient,neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis[J].Cell,1999,99(2):221-237.
[29]TISCHMEYER W,SCHICKNICK H,KRAUS M,et al.Rapamycinsensitive signalling in long-term consolidation of auditory cortex-dependent memory[J].European Journal of Neuroscience,2003,18(4):942-950.
[30]卢志杰,王文敏.雷帕霉素用于治疗神经退行性疾病研究进展[J].药物评价研究,2012(4):281-284.LU Zhi-jie,WANG Wen-min.Research advances in rapamycin against neurodegenerative disease[J].Drug Evaluation Research,2012(4):281-284.(in Chinese)
[31]CHAPUT C,ZYCHLINSKY A.Sepsis:the dark side of histones[J].Nature Medicine,2009,15(11):1245-1246.
[32]蔡靓.组蛋白在脓毒症过程中对淋巴细胞的凋亡作用及机制[D].广州:南方医科大学,2014.CAI Jing.The effect and mechanism of histones on apoptosis of lymphocytes in sepsis[D].Guangzhou:Southern Medical University,2014.(in Chinese)
[33]CHEN Y,AZAD M B,GIBSON S B.Methods for detecting autophagy and determining autophagy-induced cell death this review is one of a selection of papers published in a special issue on oxidative stress in health and disease[J].Canadian Journal of Physiology and Pharmacology,2010,88(3):285-295.
[34]洪晓丽,王洋洋,崔绍业,等.自噬减少组蛋白介导的肾小管上皮细胞凋亡[J].实用医学杂志,2017(3):350-353.HONG Xiao-li,WANG Yang-yang,CUI Shao-ye,et al.Autophagy protects against histones-mediated extracellular apoptosis in proximal tubules[J].The Journal of Practical Medicine,2017(3):350-353.(in Chinese)
[35]CHENG K W,LAHAD J P,GRAY J W,et al.Emerging role of Rab GTPases in cancer and human disease[J].Cancer Research,2005,65(7):2516-2519.
[36]AO X,ZOU L,WU Y.Regulation of autophagy by the Rab GTPase network[J].Cell Death and Differentiation,2014,21(3):348-358.
[37]AYALA J,TOUCHOT N,ZAHRAOUI A,et al.The product of rab2,a small GTP binding protein,increases neuronal adhesion,and neurite growth in vitro[J].Neuron,1990,4(5):797-805.
[38]MANGAHAS P,YU X,MILLER K G,et al.The small GTPase Rab2 functions in the removal of apoptotic cells in caenorhabditis elegans[J].The Journal of Cell Biology,2008,180(2):357-373.
[39]JAIN N,GANESH S.Emerging nexus be tween Rab GT-Pases,autophagy and neurodegeneration[J].Autophagy,2016,12(5):900-904.
[40]SOO K Y,HALLORAN M,SUNDARAMOORTHY V,et al.Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1,TDP-43 and FUS-associated ALS[J].Acta Neuropathologica,2015,130(5):679-697.
[41]BOWNES M.The regulation of the yolk protein genes,a family of sex differentiation genes in drosophila melanogaster[J].Bioessays,1994,16(10):745-752.
[42]盛岳.Rab37调控细胞增殖及自噬的分子机制研究[D].武汉:武汉大学,2014.SHENG Yue.Rab37 regulates cell proliferation and autophagy[D].Wuhan:Wuhan University,2014.(in Chinese)
[43]GUO D,ZENG L,BRODY D L,et al.Rapamycin attenuates the development of posttraumatic epilepsy in a mouse model of traumatic brain injury[J].PLoSOne,2013,8(5):e64078.
[44]LU Q,GAO L,HUANG L,et al.Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat[J].Journal of Neuroinflammation,2014,11:44.
[45]MALAGELADA C,JIN Z H,JACKSON-LEWIS V,et al.Rapamycin protects against neuron death in in vitro and in vivo models of parkinson's disease[J].Journal of Neuroscience,2010,30(3):1166-1175.
[46]SPILMAN P,PODLUTSKAYA N,HART M J,et al.Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer's disease[J].PLoS One,2010,5(4):e9979.
[47]HARRISON D E,STRONG R,SHARP Z D,et al.Rapamycin fed late in life extends lifespan in genetically heterogeneous mice[J].Nature,2009,460(7253):392-395.
[2]SA Q,OCHIAI E,TIWARI A,et al.Cutting edge:IFN-γproduced by brain-resident cells is crucial to control cerebral infection with Toxoplasma gondii[J].The Journal of Immunology,2015,195(3):796-800.
[3]PASSERI E,JONES-BRANDO L,BORDN C,et al.Infection and characterization of Toxoplasma gondii in human induced neurons from patients with brain disorders and healthy controls[J].Microbes and Infection,2016,18(2):153-158.
[4]BLANCHARD N,DUNAY I R,SCHLTER D.Persistence of Toxoplasma gondii in the central nervous system:a fine-tuned balance between the parasite,the brain and the immune system[J].Parasite Immunology,2015,37(3):150-158.
[5]FLEGR J.Effects of Toxoplasma on human behavior[J].Schizophrenia Bulletin,2007,33(3):757-760.
[6]TORREY E F,YOLKEN R H.Editors'introduction:Schizophrenia and Toxoplasmosis[J].Schizophrenia Bulletin,2007,33(3):727-728.
[7]VYAS A,SAPOLSKY R.Manipulation of host behaviour by Toxoplasma gondii:what is the minimum a proposed proximate mechanism should explain?[J].Folia Parasitologica,2010,57(2):88-94.
[8]FRIEDLANDER R M.Apoptosis and caspases in Neurodegenerative diseases[J].The New England Journal of Medicine,2003,348(14):1365-1375.
[9]EL-GUENDY N,RANGNEKAR V M.Apoptosis by Par-4 in cancer and neurodegenerative diseases[J].Experimental Cell Research,2003,283(1):51-66.
[10]LL,WANG Y,FENG W,et al.iTRAQ-based differential proteomic analysis in Mongolian gerbil brainschronically infected with Toxoplasma gondii[J].Journal of Proteomics,2017,160:74-83.
[11]ALLAM R,SCHERBAUM C R,DARISIPUDI M N,et al.Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4[J].Journal of the American Society of Nephrology,2012,23(8):1375-1388.
[12]XU J,ZHANG X,PELAYO R,et al.Extracellular histones are major mediators of death in sepsis[J].Nature Medicine,2009,15(11):1318-1321.
[13]LIU Z,NI S,CHEN G,et al.Histones-mediated lymphocyte apoptosis during sepsis is dependent on p38 phosphorylation and mitochondrial permeability transition[J].PLoS One,2013,8(10):e77131.
[14]STENMARK H,OLKKONEN V M.The Rab GTPase family[J].Genome Biology,2001,2(5):S3007.
[15]MOHRMANN K,VAN DER SLUIJS P.Regulation of membrane transport through the endocytic pathway by rab GTPases[J].Molecular Membrane Biology,1999,16(1):81-87.
[16]STENMARK H.Rab GTPases as coordinators of vesicle traffic[J].Nature Reviews Molecular Cell Biology,2009,10(8):513-525.
[17]KHOKHA R,MURTHY A,WEISS A.Metalloproteinases and their natural inhibitors in inflammation and immunity[J].Nature Reviews Immunology,2013,13(9):649-665.
[18]陈华萍,陈旭昕,董伟杰,等.Rab26诱导宫颈癌HeLa细胞凋亡并抑制其迁移[J].第三军医大学学报,2016(24):2576-2579.CHEN Hua-ping,CHEN Xu-xin,DONG Wei-jie,et al.Rab26 induces apoptosis and suppresses migration of He La cells[J].Journal of Third Military Medical University,2016(24):2576-2579.(in Chinese)
[19]李小山,李国利,冯晓灵,等.过表达Rab27B体外促进人胃癌细胞系MKN-45凋亡并抑制其增殖[J].基础医学与临床,2016(7):976-980.LI Xiao-shan,LI Guo-li,FENG Xiao-ling,et al.Overexpressed Rab27B induces apoptosis and inhibits proliferation of MKN45 cell line in vitro[J].Basic&Clinical Medicine,2016(7):976-980.(in Chinese)
[20]ROHDE J R,CARDENAS M E.The tor pathway regulates gene expression by linking nutrient sensing to histone acetylation[J].Molecular and Cel lular Biology,2003,23(2):629-635.
[21]YU Z,KONE B C.Targeted histone H4 acetylation via phosphoinositide 3-kinase-and p70s6-kinase-dependent pathways inhibits iNOS induction in mesangial cells[J].American Journal of Physiolo gyRenal Physiology,2006,290(2):496-502.
[22]DURAN R V,HALL M N.Regulation of TOR by small GT-Pases[J].EMBO Reports,2012,13(2):121-128.
[23]LI L,KIM E,YUAN H,et al.Regulation of mTORC1 by the Rab and Arf GTPases[J].Journal of Biological Chemistry,2010,285(26):19705-19709.
[24]MATSUI T,FUKUDA M.Rab12.regulates mTORC1 activity and autophagy through controlling the degradation of amino-acid transporter PAT4[J].EMBOReports,2013,14(5):450-457.
[25]CARGNELLO M,TCHERKEZIAN J,ROUX P P.The expanding role of mTOR in cancer cell growth and proliferation[J].Mutagenesis,2015,30(2):169-176.
[26]CACCAMO A,MAJUMDER S,RICHARDSON A,et al.Molecular interplay between mammalian target of rapamycin(mTOR),amyloid-β,and Tau[J].Journal of Biological Chemistry,2010,285(17):13107-13120.
[27]CACCAMO A,MALDONADO M A,MAJUMDER S,et al.Naturally secreted amyloid-βincreases mammalian target of rapamycin(mTOR)activity via a PRAS40-mediated mechanism[J].Journal of Biological Chemistry,2011,286(11):8924-8932.
[28]CASADIO A,MARTIN K C,GIUSTETTO M,et al.A transient,neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis[J].Cell,1999,99(2):221-237.
[29]TISCHMEYER W,SCHICKNICK H,KRAUS M,et al.Rapamycinsensitive signalling in long-term consolidation of auditory cortex-dependent memory[J].European Journal of Neuroscience,2003,18(4):942-950.
[30]卢志杰,王文敏.雷帕霉素用于治疗神经退行性疾病研究进展[J].药物评价研究,2012(4):281-284.LU Zhi-jie,WANG Wen-min.Research advances in rapamycin against neurodegenerative disease[J].Drug Evaluation Research,2012(4):281-284.(in Chinese)
[31]CHAPUT C,ZYCHLINSKY A.Sepsis:the dark side of histones[J].Nature Medicine,2009,15(11):1245-1246.
[32]蔡靓.组蛋白在脓毒症过程中对淋巴细胞的凋亡作用及机制[D].广州:南方医科大学,2014.CAI Jing.The effect and mechanism of histones on apoptosis of lymphocytes in sepsis[D].Guangzhou:Southern Medical University,2014.(in Chinese)
[33]CHEN Y,AZAD M B,GIBSON S B.Methods for detecting autophagy and determining autophagy-induced cell death this review is one of a selection of papers published in a special issue on oxidative stress in health and disease[J].Canadian Journal of Physiology and Pharmacology,2010,88(3):285-295.
[34]洪晓丽,王洋洋,崔绍业,等.自噬减少组蛋白介导的肾小管上皮细胞凋亡[J].实用医学杂志,2017(3):350-353.HONG Xiao-li,WANG Yang-yang,CUI Shao-ye,et al.Autophagy protects against histones-mediated extracellular apoptosis in proximal tubules[J].The Journal of Practical Medicine,2017(3):350-353.(in Chinese)
[35]CHENG K W,LAHAD J P,GRAY J W,et al.Emerging role of Rab GTPases in cancer and human disease[J].Cancer Research,2005,65(7):2516-2519.
[36]AO X,ZOU L,WU Y.Regulation of autophagy by the Rab GTPase network[J].Cell Death and Differentiation,2014,21(3):348-358.
[37]AYALA J,TOUCHOT N,ZAHRAOUI A,et al.The product of rab2,a small GTP binding protein,increases neuronal adhesion,and neurite growth in vitro[J].Neuron,1990,4(5):797-805.
[38]MANGAHAS P,YU X,MILLER K G,et al.The small GTPase Rab2 functions in the removal of apoptotic cells in caenorhabditis elegans[J].The Journal of Cell Biology,2008,180(2):357-373.
[39]JAIN N,GANESH S.Emerging nexus be tween Rab GT-Pases,autophagy and neurodegeneration[J].Autophagy,2016,12(5):900-904.
[40]SOO K Y,HALLORAN M,SUNDARAMOORTHY V,et al.Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1,TDP-43 and FUS-associated ALS[J].Acta Neuropathologica,2015,130(5):679-697.
[41]BOWNES M.The regulation of the yolk protein genes,a family of sex differentiation genes in drosophila melanogaster[J].Bioessays,1994,16(10):745-752.
[42]盛岳.Rab37调控细胞增殖及自噬的分子机制研究[D].武汉:武汉大学,2014.SHENG Yue.Rab37 regulates cell proliferation and autophagy[D].Wuhan:Wuhan University,2014.(in Chinese)
[43]GUO D,ZENG L,BRODY D L,et al.Rapamycin attenuates the development of posttraumatic epilepsy in a mouse model of traumatic brain injury[J].PLoSOne,2013,8(5):e64078.
[44]LU Q,GAO L,HUANG L,et al.Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat[J].Journal of Neuroinflammation,2014,11:44.
[45]MALAGELADA C,JIN Z H,JACKSON-LEWIS V,et al.Rapamycin protects against neuron death in in vitro and in vivo models of parkinson's disease[J].Journal of Neuroscience,2010,30(3):1166-1175.
[46]SPILMAN P,PODLUTSKAYA N,HART M J,et al.Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer's disease[J].PLoS One,2010,5(4):e9979.
[47]HARRISON D E,STRONG R,SHARP Z D,et al.Rapamycin fed late in life extends lifespan in genetically heterogeneous mice[J].Nature,2009,460(7253):392-395.