肠道病毒A71型致人扁桃体上皮细胞系UT-SCC-60B凋亡和S期阻滞的研究
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摘要
肠道病毒A71型(Enterovirus A71,EV-A71)是手足口病的重要病原体,为研究EV-A71感染人扁桃体上皮细胞后对细胞凋亡和细胞周期的影响,确定ERK1/2、JNK1/2、PI3K/Akt和含半胱氨酸的天冬氨酸蛋白水解酶(Cysteinyl aspartate specific proteinase,Caspase)的作用,本文以人扁桃体上皮细胞系UT-SCC-60B为细胞模型,CCK-8试剂盒检测EV-A71对UT-SCC-60B的抑制率、流式细胞仪检测EV-A71感染组和抑制剂处理组的凋亡和细胞周期、Caspase活力检测试剂盒测定Caspase-3,Caspase-8,Caspase-9活力。EV-A71以感染剂量和感染时间依赖方式抑制UT-SCC-60B增殖;EV-A71感染致UT-SCC-60B发生细胞凋亡,抑制ERK1/2、JNK1/2和PI3K/Akt能够降低UT-SCC-60B细胞凋亡比例;EV-A71感染UT-SCC-60B后发生S期阻滞,抑制ERK1/2、JNK1/2、PI3K/Akt和Caspase阻止UT-SCC-60B发生S期阻滞;EV-A71感染UT-SCC-60B能够活化Caspase-3,Caspase-8,Caspase-9且ERK1/2、JNK1/2和PI3K/Akt调控Caspase-3,Caspase-8,Caspase-9活力。因此,EV-A71能够导致人扁桃体上皮细胞UT-SCC-60B发生凋亡和S期阻滞,并且ERK1/2、JNK1/2、PI3K/Akt和Caspase参与凋亡和S期阻滞的调控。
Enterovirus-A71(EV-A71) is the major pathogen of hand, foot and mouth disease.To investigate the apoptosis and cell cycle in EV-A71-infected human tonsillar epithelial cells and determine the roles of ERK1/2,JNK1/2, PI3 K/Akt and Caspase, the human tonsillar epithelial cell line UT-SCC-60B was chosen as a model.Cell Counting kit-8(CCK-8) was used to detect the inhibition of UT-SCC-60B cells caused by EV-A71 infection. Apoptosis and cell cycles were detected using flow cytometry in EV-A71-infected groups and inhibitortreated groups. Activities of Caspase-3, Caspase-8 and Caspase-9 were measured using Caspase detection kits.Results showed that proliferation of UT-SCC-60B cells was inhibited by EV-A71 in dose-and time-dependent manners. UT-SCC-60B cells underwent apoptosis upon EV-A71 infection,and the apoptosis rate was decreased by inhibition of the ERK1/2, JNK1/2 and PI3 K/Akt pathways. EV-A71 infection induced S-phase arrest in UT-SCC-60B cells, and the percentage of the S phase also declined upon inhibition of ERK1/2, JNK1/2,PI3 K/Akt and Caspase expression. EV-A71 induced the activities of Caspase-3, Caspase-8 and Caspase-9, and these activities were regulated by the ERK1/2, JNK1/2 and PI3 K/Ak1 pathways. In conclusion, apoptosis and S-phase arrest were induced in EV-A71-infected UT-SCC-60B cells, and ERK1/2, JNK1/2, PI3 K/Akt and Caspase pathways regulated apoptosis and cell-cycle arrest.
Enterovirus-A71(EV-A71) is the major pathogen of hand, foot and mouth disease.To investigate the apoptosis and cell cycle in EV-A71-infected human tonsillar epithelial cells and determine the roles of ERK1/2,JNK1/2, PI3 K/Akt and Caspase, the human tonsillar epithelial cell line UT-SCC-60B was chosen as a model.Cell Counting kit-8(CCK-8) was used to detect the inhibition of UT-SCC-60B cells caused by EV-A71 infection. Apoptosis and cell cycles were detected using flow cytometry in EV-A71-infected groups and inhibitortreated groups. Activities of Caspase-3, Caspase-8 and Caspase-9 were measured using Caspase detection kits.Results showed that proliferation of UT-SCC-60B cells was inhibited by EV-A71 in dose-and time-dependent manners. UT-SCC-60B cells underwent apoptosis upon EV-A71 infection,and the apoptosis rate was decreased by inhibition of the ERK1/2, JNK1/2 and PI3 K/Akt pathways. EV-A71 infection induced S-phase arrest in UT-SCC-60B cells, and the percentage of the S phase also declined upon inhibition of ERK1/2, JNK1/2,PI3 K/Akt and Caspase expression. EV-A71 induced the activities of Caspase-3, Caspase-8 and Caspase-9, and these activities were regulated by the ERK1/2, JNK1/2 and PI3 K/Ak1 pathways. In conclusion, apoptosis and S-phase arrest were induced in EV-A71-infected UT-SCC-60B cells, and ERK1/2, JNK1/2, PI3 K/Akt and Caspase pathways regulated apoptosis and cell-cycle arrest.
引文
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[13] Tung W H, Lee I T, Hsieh H L, Yang C M, EV71 induces COX-2 expression via c-Src/PDGFR/PI3K/Akt/p42/p44 MAPK/AP-1 and NF-kappaB in rat brain astrocytes[J]. J Cell Physiol,2010,224(2):376-386.
[14] Wong W R, Chen Y Y, Yang S M, Chen Y L, Horng J T. Phosphorylation of PI3K/Akt and MAPK/ERK in an early entry step of enterovirus 71[J]. Life Sci, 2005,78(1):82-90.
[15] Zhang H,Cong H, Song L, Tien P. The nuclear protein Sam68 is redistributed to the cytoplasm and is involved in PI3K/Akt activation during EV71 infection[J]. Virus Res,2014,180:1-11.
[16] Zhang H,Li F,Pan Z,Wu Z,Wang Y,Cui Y. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection[J]. Virus Res, 2014, 192:74-84.
[17] Shi W, Hou X, Li X, Peng H, Shi M, Jiang Q, Liu X,Ji Y,Yao Y, He C, Lei X. Differential apoptosis gene expressions of rhabdomyosarcoma cells in response to enterovirus 71 infection[J]. BMC Infect Dis, 2012,12:327.
[18] Larsen B D, Sorensen C S. The caspase-activated DNase:apoptosis and beyond[J]. FEBS J,2017,284(8):1160-1170.
[19] Howe J A,Mymryk J S,Egan C,Branton P E,Bayley S T. Retinoblastoma growth suppressor and a 300-kDa protein appear to regulate cellular DNA synthesis[J].Proc Natl Acad Sci U S A,1990,87(15):5883-5887.
[20] Werness B A, Levine A J, Howley P M. Association of human papillomavirus types 16 and 18 E6 proteins withp53[J]. Science, 1990,248(4951):76-79.
[21] Song F, Yu X,Zhong T,Wang Z,Meng X,Li Z,Zhang S,Huo W,Liu X,Zhang Y,Zhang W,Yu J.Caspase-3 inhibition attenuates the cytopathic effects of ev71 infection[J]. Front Microbiol,2018,9:817.
[2] Jiang M,Wei D,Ou WL,Li K X,Luo D Z,Li Y Q,Chen E,Nong G M. Autopsy findings in children with hand,foot,and mouth disease[J]. N Engl J Med,2012,367(1):91-92.
[3] Zhang Y,Zhu Z,Yang W,Ren J,Tan X,Wang Y,Mao N,Xu S,Zhu S,Cui A,Zhang Y,Yan D,Li Q,Dong X,Zhang J,Zhao Y,Wan J,Feng Z, Sun J,Wang S, Li D, Xu W. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China[J]. Virol J,2010,7:94.
[4] Zhang Y,Tan X J,Wang H Y,Yan D M,Zhu S L,Wang D Y,Ji F,Wang X J,Gao Y J,Chen L,An H Q, Li D X, Wang S W, Xu A Q, Wang Z J, Xu W B.An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong,China[J]. J Clin Virol, 2009,44(4):262-267.
[5] Hu Y Q, Xie G C, Li D D, Pang LL, Xie J, Wang P,Chen Y,Yang J,Cheng W X,Zhang Q,Jin Y,Duan Z J. Prevalence of coxsackievirus A6 and Enterovirus 71in hand, foot and mouth disease in Nanjing,China in2013[J]. Pediatr Infect Dis J,2015,34(9):951-957.
[6] Ooi M H,Solomon T,Podin Y,Mohan A,Akin W,Yusuf M A, del Sel S, Kontol K M, Lai B F, Clear D,Chieng C H,Blake E,Perera D,Wong S C,Cardosa J. Evaluation of different clinical sample types in diagnosis of human enterovirus 71-associated hand-foot-andmouth disease[J]. J Clin Microbiol, 2007,45(6):1858-1866.
[7] He Y, Ong K C, Gao Z, Zhao X, Anderson V M, McNutt M A, Wong K T, Lu M. Tonsillar crypt epithelium is an important extra-central nervous system site forviral replication in EV71 encephalomyelitis[J].Am J Pathol,2014,184(3):714-720.
[8] Ong K C,Wong K T. Understanding enterovirus 71neuropathogenesis and its impact on other neurotropic enteroviruses[J]. Brain Pathol,2015,25(5):614-624.
[9] Xi X,Zhang X,Wang B,Wang T,Wang J,Huang H,Wang J,Jin Q,Zhao Z. The interplays between autophagy and apoptosis induced by enterovirus 71[J/OL].PLoS One,2013,8(2):e56966.
[10] Yu J, Zhang L, Ren P, Zhong T, Li Z, Wang Z, Li J,Liu X, Zhao K, Zhang W, Yu X F. Enterovirus 71 mediates cell cycle arrest in S phase through non-structural protein 3D[J]. Cell Cycle, 2015,14(3):425-436.
[11] Du X,Wang H,Xu F,Huang Y,Liu Z,Liu T. Enterovirus 71 induces apoptosis of SHSY5Y human neuroblastoma cells through stimulation of endogenous microRNA let-7b expression[J]. Mol Med Rep,2015, 12(1):953-959.
[12] Xie G C, Guo N J, Grenman R, Wang H, Wang Y,Vuorenmma M, Zhang Q, Zhang S, Li H Y, Pang L L,Li D D,Jin M,Sun X M,Kong X Y,Duan Z J.Susceptibility of human tonsillar epithelial cells to enterovirus 71 with normal cytokine response[J]. Virology,2016,494:108-118.
[13] Tung W H, Lee I T, Hsieh H L, Yang C M, EV71 induces COX-2 expression via c-Src/PDGFR/PI3K/Akt/p42/p44 MAPK/AP-1 and NF-kappaB in rat brain astrocytes[J]. J Cell Physiol,2010,224(2):376-386.
[14] Wong W R, Chen Y Y, Yang S M, Chen Y L, Horng J T. Phosphorylation of PI3K/Akt and MAPK/ERK in an early entry step of enterovirus 71[J]. Life Sci, 2005,78(1):82-90.
[15] Zhang H,Cong H, Song L, Tien P. The nuclear protein Sam68 is redistributed to the cytoplasm and is involved in PI3K/Akt activation during EV71 infection[J]. Virus Res,2014,180:1-11.
[16] Zhang H,Li F,Pan Z,Wu Z,Wang Y,Cui Y. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection[J]. Virus Res, 2014, 192:74-84.
[17] Shi W, Hou X, Li X, Peng H, Shi M, Jiang Q, Liu X,Ji Y,Yao Y, He C, Lei X. Differential apoptosis gene expressions of rhabdomyosarcoma cells in response to enterovirus 71 infection[J]. BMC Infect Dis, 2012,12:327.
[18] Larsen B D, Sorensen C S. The caspase-activated DNase:apoptosis and beyond[J]. FEBS J,2017,284(8):1160-1170.
[19] Howe J A,Mymryk J S,Egan C,Branton P E,Bayley S T. Retinoblastoma growth suppressor and a 300-kDa protein appear to regulate cellular DNA synthesis[J].Proc Natl Acad Sci U S A,1990,87(15):5883-5887.
[20] Werness B A, Levine A J, Howley P M. Association of human papillomavirus types 16 and 18 E6 proteins withp53[J]. Science, 1990,248(4951):76-79.
[21] Song F, Yu X,Zhong T,Wang Z,Meng X,Li Z,Zhang S,Huo W,Liu X,Zhang Y,Zhang W,Yu J.Caspase-3 inhibition attenuates the cytopathic effects of ev71 infection[J]. Front Microbiol,2018,9:817.