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研究生：	廖紹含 Liao, Shao-Han
論文名稱：	探討含雙色胺酸功能區氧化還原酶與腸病毒71型感染症之交互調控作用 The interplay of WW domain-containing oxidoreductase with enterovirus 71
指導教授：	徐麗君 Hsu, Li-Jin
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	66
中文關鍵詞：	含雙色胺酸功能區氧化還原酶、腸病毒71型
外文關鍵詞：	WWOX, EV71
相關次數：	點閱：109 下載：0
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含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase)，又名為WWOX或小鼠WOX1，為一種抑癌蛋白質。先前研究指出，WWOX蛋白質參與多種訊息傳遞路徑，以發揮抑癌功能、調控細胞凋亡、參與胚胎發育、以及影響神經性疾病的發展。先前亦有研究顯示，WWOX蛋白質與EB病毒 (Epstein-Barr virus) 和人類T細胞白血病毒第1型 (human T cell leukemia virus, HTLV-1) 誘導的癌症調控機制有關。然而，現今尚未有研究證明 WWOX蛋白質是否調控哺乳類細胞受到腸病毒71型 (enterovirus 71; EV71) 感染的作用。為了進一步探討WWOX在EV71感染下可能扮演的角色，我們利用lentiviral shRNA，將人類橫紋肌肉瘤細胞 (human rhabdomyosarcoma, RD) 內WWOX的表現抑制下來。有趣的是，與控制組細胞相較之下，WWOX蛋白質表現量很低的RD細胞在受到EV71感染後，細胞內EV71的表現量較高。 RD細胞中WWOX蛋白質表現量減少時，並不會改變EV71受器SCARB2與Nucleolin的表現量，病毒結合到細胞表面的情形也沒有受到影響。 Wwox基因剔除小鼠 (knockout, Wwox-/-) 在感染EV71後，比起野生種 (wild-type, Wwox+/+)出現較高死亡率。綜合以上，我們發現WWOX蛋白質可調控EV71在動物體內及細胞內的感染情形。

WW domain-containing oxidoreductase, designated WWOX or murine WOX1, is a candidate tumor suppressor protein. WWOX has been suggested to be involved in many signaling pathways that regulate tumor suppression, cell death, embryonic development and neuronal diseases. Previous studies have suggested that WWOX is associated with cancer progression induced by Epstein-Barr virus and human T cell leukemia virus. However, whether WWOX controls the infection of mammalian cells by enterovirus 71 (EV71) is still unknown. To investigate the possible role of WWOX in EV71 infection, lentiviral shRNA-mediated knockdown of WWOX expression was used in human rhabdomyosarcoma (RD) cells. Interestingly, higher viral levels were detected in WWOX-knockdown RD cells than the controls after EV71 infection. Comparable expression levels of cellular receptors for EV71, SCARB2 and Nucleolin, were detected in WWOX-knockdown and control RD cells. Knockdown of WWOX expression in RD cells did not affect the binding of EV71 to the cell surface. Wwox gene knockout mice exhibited higher mortality rates than did wild-type littermates after EV71 infection. Taken together, we have demonstrated that WWOX regulates EV71 infection both in vitro and in vivo.

中文摘要	I
Abstract	II
致謝	III
Contents	IV 
Figure index	VII
Introduction	1
WW domain-containing oxidoreductase	1
Functions of WWOX protein	1
Relationship between WWOX protein and EBV infection	2
WWOX decreases NF-B activation in HTLV-1 infection	2
Enterovirus 71 (EV71)	3
Diseases caused by EV71 infection	3
Transmission and life cycle of EV71	4
EV71 induces autophagy to promote its replication	4
The signaling pathways that regulate EV71 replication in host cells	5
Mouse models for EV71 infection	5
Materials and Methods	7
A. Materials	7
A-1 Cell lines	7
A-2 Chemicals and reagents	7
A-3 Kits	10
A-4 Antibodies	10
A-5 Consumables	11
A-6 Instruments	12
B. Methods	13
B-1 Cell culture	13
B-2 Virus	13
B-3 Plaque assay	14
B-4 WWOX knockdown in cell lines	15
B-5 Virus binding assay: EV71 binding rate at 4℃	16
B-6 Virus binding assay: ELISA assay	17
B-7 Protein extraction	19
B-8 Protein quantification	20
B-9 Generation and extraction of EV71	21
B-10 RNA extraction	22
B-11 Reverse transcription-polymerase chain reaction (RT-PCR)	23
B-13 Plasmid DNA purification	25
B-14 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting	27
B-15 Hematoxylin and eosin (HE) staining	29
B-16 Immunofluorescence (IF) staining	30
B-17 EV71 infection of Wwox gene knockout mice	31
B-18 Organ dissemination	32
Results	33
EV71 infection upregulates WWOX protein expression in human RD cells	33
WWOX protein impedes in EV71 replication in RD cells	33
EV71 binds equally well to shLuc and shWWOX RD cells	34
Knockdown of WWOX expression enhances endocytosis of EV71 in RD cells	35
Wwox protects mice from lethal infection with EV71	36
Wwox expression decreases the viral loads in multiple organs of EV71-infected mice	37
EV71 infection causes higher mortality rate in T-cell specific Wwox knockout mice	38
Discussion	39
The role of WWOX protein in vitro and in vivo	39
WWOX affects the entry of EV71 into cells	39
Active IRES of WWOX may compete for the initiation factors to inhibit EV71 translation	40
WWOX reduces autophagy to suppress EV71 replication	41
The possible WWOX-mediated signaling pathways for regulating EV71 replication	41
Conclusion	43
References	44
Figures……………………………………………………………………………………..48
Figure 1………………………………………………………………………….………….....48
Figure 2………………………………………………………………………………………..49
Figure 3………………………………………………………………………………………..50
Figure 4………………………………………………………………………………………..52
Figure 5………………………………………………………………………………………..53
Figure 6………………………………………………………………………………………..56
Figure 7………………………………………………………………………………………..57
Figure 8………………………………………………………………………………………..59
Figure 9………………………………………………………………………………………..60
Figure 10………………………………………………………………………………………62
Figure 11………………………………………………………………………………………63
Figure 12………………………………………………………………………………………65
                                    

[1] A. K. Bednarek, K. J. Laflin, R. L. Daniel, Q. Liao, K. A. Hawkins, and C. M. Aldaz, "WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer," Cancer Res, vol. 60, pp. 2140-5, Apr 15 2000.
[2] N. S. Chang, L. J. Hsu, Y. S. Lin, F. J. Lai, and H. M. Sheu, "WW domain-containing oxidoreductase: a candidate tumor suppressor," Trends Mol Med, vol. 13, pp. 12-22, Jan 2007.
[3] H. R. Qin, D. Iliopoulos, S. Semba, M. Fabbri, T. Druck, S. Volinia, et al., "A role for the WWOX gene in prostate cancer," Cancer Res, vol. 66, pp. 6477-81, Jul 1 2006.
[4] J. P. Cantor, D. Iliopoulos, A. S. Rao, T. Druck, S. Semba, S. Y. Han, et al., "Epigenetic modulation of endogenous tumor suppressor expression in lung cancer xenografts suppresses tumorigenicity," Int J Cancer, vol. 120, pp. 24-31, Jan 1 2007.
[5] R. I. Aqeilan, M. Q. Hassan, A. de Bruin, J. P. Hagan, S. Volinia, T. Palumbo, et al., "The WWOX tumor suppressor is essential for postnatal survival and normal bone metabolism," J Biol Chem, vol. 283, pp. 21629-39, Aug 1 2008.
[6] C. I. Sze, M. Su, S. Pugazhenthi, P. Jambal, L. J. Hsu, J. Heath, et al., "Down-regulation of WW domain-containing oxidoreductase induces Tau phosphorylation in vitro. A potential role in Alzheimer's disease," J Biol Chem, vol. 279, pp. 30498-506, Jul 16 2004.
[7] M. Mallaret, M. Synofzik, J. Lee, C. A. Sagum, M. Mahajnah, R. Sharkia, et al., "The tumour suppressor gene WWOX is mutated in autosomal recessive cerebellar ataxia with epilepsy and mental retardation," Brain, vol. 137, pp. 411-9, Feb 2014.
[8] C. W. Tsai, F. J. Lai, H. M. Sheu, Y. S. Lin, T. H. Chang, M. S. Jan, et al., "WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma," Cell Death Dis, vol. 4, p. e792, 2013.
[9] J. Fu, Z. Qu, P. Yan, C. Ishikawa, R. I. Aqeilan, A. B. Rabson, et al., "The tumor suppressor gene WWOX links the canonical and noncanonical NF-kappaB pathways in HTLV-I Tax-mediated tumorigenesis," Blood, vol. 117, pp. 1652-61, Feb 3 2011.
[10] Y. Y. Lan, S. Y. Wu, H. C. Lai, N. S. Chang, F. H. Chang, M. H. Tsai, et al., "WW domain-containing oxidoreductase is involved in upregulation of matrix metalloproteinase 9 by Epstein-Barr virus latent membrane protein 2A," Biochem Biophys Res Commun, vol. 436, pp. 672-6, Jul 12 2013.
[11] E. W. Harhaj and N. S. Harhaj, "Mechanisms of persistent NF-kappaB activation by HTLV-I tax," IUBMB Life, vol. 57, pp. 83-91, Feb 2005.
[12] T. Solomon, P. Lewthwaite, D. Perera, M. J. Cardosa, P. McMinn, and M. H. Ooi, "Virology, epidemiology, pathogenesis, and control of enterovirus 71," Lancet Infect Dis, vol. 10, pp. 778-90, Nov 2010.
[13] J. M. Wu, J. N. Wang, Y. C. Tsai, C. C. Liu, C. C. Huang, Y. J. Chen, et al., "Cardiopulmonary manifestations of fulminant enterovirus 71 infection," Pediatrics, vol. 109, pp. E26-, Feb 2002.
[14] L. C. Lum, K. T. Wong, S. K. Lam, K. B. Chua, and A. Y. Goh, "Neurogenic pulmonary oedema and enterovirus 71 encephalomyelitis," Lancet, vol. 352, p. 1391, Oct 24 1998.
[15] S. Yamayoshi, S. Ohka, K. Fujii, and S. Koike, "Functional comparison of SCARB2 and PSGL1 as receptors for enterovirus 71," J Virol, vol. 87, pp. 3335-47, Mar 2013.
[16] S. Yamayoshi, K. Fujii, and S. Koike, "Scavenger receptor b2 as a receptor for hand, foot, and mouth disease and severe neurological diseases," Front Microbiol, vol. 3, p. 32, 2012.
[17] R. D. Cummings, "Structure and function of the selectin ligand PSGL-1," Braz J Med Biol Res, vol. 32, pp. 519-28, May 1999.
[18] H. Y. Lin, Y. T. Yang, S. L. Yu, K. N. Hsiao, C. C. Liu, C. Sia, et al., "Caveolar endocytosis is required for human PSGL-1-mediated enterovirus 71 infection," J Virol, vol. 87, pp. 9064-76, Aug 2013.
[19] Y. Sun, Y. Guo, and Z. Lou, "Formation and working mechanism of the picornavirus VPg uridylylation complex," Curr Opin Virol, vol. 9C, pp. 24-30, Sep 18 2014.
[20] J. Y. Lin, T. C. Chen, K. F. Weng, S. C. Chang, L. L. Chen, and S. R. Shih, "Viral and host proteins involved in picornavirus life cycle," J Biomed Sci, vol. 16, p. 103, 2009.
[21] Y. R. Lee, P. S. Wang, J. R. Wang, and H. S. Liu, "Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model," J Biomed Sci, vol. 21, p. 80, 2014.
[22] W. H. Tung, H. L. Hsieh, and C. M. Yang, "Enterovirus 71 induces COX-2 expression via MAPKs, NF-kappaB, and AP-1 in SK-N-SH cells: Role of PGE(2) in viral replication," Cell Signal, vol. 22, pp. 234-46, Feb 2010.
[23] W. H. Tung, H. L. Hsieh, I. T. Lee, and C. M. Yang, "Enterovirus 71 modulates a COX-2/PGE2/cAMP-dependent viral replication in human neuroblastoma cells: role of the c-Src/EGFR/p42/p44 MAPK/CREB signaling pathway," J Cell Biochem, vol. 112, pp. 559-70, Feb 2011.
[24] W. X. Khong, B. Yan, H. Yeo, E. L. Tan, J. J. Lee, J. K. Ng, et al., "A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection," J Virol, vol. 86, pp. 2121-31, Feb 2012.
[25] Y. F. Wang, C. T. Chou, H. Y. Lei, C. C. Liu, S. M. Wang, J. J. Yan, et al., "A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection," J Virol, vol. 78, pp. 7916-24, Aug 2004.
[26] J. Lu, Y. Q. He, L. N. Yi, H. Zan, H. F. Kung, and M. L. He, "Viral kinetics of enterovirus 71 in human abdomyosarcoma cells," World J Gastroenterol, vol. 17, pp. 4135-42, Sep 28 2011.
[27] S. Bose, M. Basu, and A. K. Banerjee, "Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells," J Virol, vol. 78, pp. 8146-58, Aug 2004.
[28] C. Haolong, N. Du, T. Hongchao, Y. Yang, Z. Wei, Z. Hua, et al., "Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells," PLoS One, vol. 8, p. e73900, 2013.
[29] T. Y. Lin, S. H. Hsia, Y. C. Huang, C. T. Wu, and L. Y. Chang, "Proinflammatory cytokine reactions in enterovirus 71 infections of the central nervous system," Clin Infect Dis, vol. 36, pp. 269-74, Feb 1 2003.
[30] J. H. Ludes-Meyers, H. Kil, A. K. Bednarek, J. Drake, M. T. Bedford, and C. M. Aldaz, "WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins," Oncogene, vol. 23, pp. 5049-55, Jun 24 2004.
[31] K. Kosla, M. Nowakowska, K. Pospiech, and A. K. Bednarek, "WWOX modulates the gene expression profile in the T98G glioblastoma cell line rendering its phenotype less malignant," Oncol Rep, vol. 32, pp. 1362-8, Oct 2014.
[32] J. Samaj, F. Baluska, B. Voigt, M. Schlicht, D. Volkmann, and D. Menzel, "Endocytosis, actin cytoskeleton, and signaling," Plant Physiol, vol. 135, pp. 1150-61, Jul 2004.
[33] E. Smythe and K. R. Ayscough, "Actin regulation in endocytosis," J Cell Sci, vol. 119, pp. 4589-98, Nov 15 2006.
[34] S. R. Thompson and P. Sarnow, "Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved," Virology, vol. 315, pp. 259-266, 2003.
[35] S. C. Huang, C. L. Chang, P. S. Wang, Y. Tsai, and H. S. Liu, "Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication," J Med Virol, vol. 81, pp. 1241-52, Jul 2009.
[36] M. Y. Li, F. J. Lai, L. J. Hsu, C. P. Lo, C. L. Cheng, S. R. Lin, et al., "Dramatic co-activation of WWOX/WOX1 with CREB and NF-kappaB in delayed loss of small dorsal root ganglion neurons upon sciatic nerve transection in rats," PLoS One, vol. 4, p. e7820, 2009.
[37] Z. Zheng, H. Li, Z. Zhang, J. Meng, D. Mao, B. Bai, et al., "Enterovirus 71 2C protein inhibits TNF-alpha-mediated activation of NF-kappaB by suppressing IkappaB kinase beta phosphorylation," J Immunol, vol. 187, pp. 2202-12, Sep 1 2011.

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