子宮頸癌為我國婦女十大死因之一，研究中發現人類乳突病毒 (hunman papillomavirus, HPV)18和16型產生的致癌蛋白E6和E7為最主要導致子宮頸細胞癌化的主要因子。E6和E7致癌蛋白最主要是利用和參與細胞生長有關的p53以及視網膜母細胞瘤 (retinoblastoma) 抑癌蛋白的結合進而抑制其蛋白的表現而使細胞產生癌化現象。核醣核酸干擾作用是一種只在真核生物發現的機制。最主要是利用小片段雙股的核醣核酸序列在細胞質內和其配對的訊息核醣核酸進行專一性鍵結而導致訊息核醣核酸降解的作用。在之前一些相關研究中，指出可以利用誘導核醣核酸干擾作用產生來抑制人類乳突病毒的轉譯作用進而達到治療子宮頸癌。因此在我們的研究中，我企圖利用之前實驗室所發展出篩選有效小片段干擾核醣核酸的平台，針對人類乳突病毒找出有效的小片段干擾核醣核酸。我們已經利用此平台針對16以及18型病毒分別找出兩個有效的標的序列。我們希望之後在將表現小片段干擾核醣核酸的載體送入細胞時，能夠如預期的有效將E6以及E7致癌蛋白的表現量降低。而使得癌細胞因為抑癌因子的回復產生細胞凋亡的現象。
　　在1969年加州腦炎嬰兒病例中首次分離出腸病毒71 (Enterovirus 71, EV71)，分類上屬小核醣核酸病毒屬腸病毒種，腸病毒71型被認為是造成手足口病(hand、foot、mouth disease, HFMD)，與一些地區性腦脊髓炎 (encephalomyelitis) 的主因，在1998年台灣曾爆發大規模流行，並且至少有超過9000個被感染的病例，其中更有55個兒童死於腦中樞神經系統的病變。IRES (internal ribosomal entry site) 一般認為對於腸病毒進行轉譯是非常重要的。因此本實驗室企圖利用有效的小片段干擾核醣核酸序列來抑制腸病毒71台灣型4643的IRES (internal ribosomal entry site) 作用進而抑制腸病毒感染。我們已經利用之前所建立的篩選平台找到有效的標的序列。更進一步利用此序列針對IRES (internal ribosomal entry site) 整段基因的表現載體進行沉默作用，發現其中一個標的序列對於沉默IRES (internal ribosomal entry site) 具有相當大的效果。我們將會把篩選出的有效標的序列送入受感染的細胞中，希望可以真正有效的降低腸病毒的感染能力。

Human papillomavirus, causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance. The E6 and E7 oncoproteins of high-risk HPV, particularly HPV 18 and 16, bind respectively to the p53 and Retinoblastoma (Rb) tumor suppressor proteins, which are involved in the regulation of cell growth. RNA interference (RNAi) is a sequence-specific RNA degradation process in the cytoplasm of eukaryotic cells that is induced by double-stranded RNA. Inhibition of HPV expression by means of RNAi has been reported in many papers. In this study, we have screened the effective siRNA/shRNA by siRNA validation system. By transfection of these shRNA to cervical cancer cell, we found that E6 and E7 shRNA induced accumulation of p53 by decreasing mRNA encoding E6 and E7 protein. We thought that HPV E6 and E7 siRNA as a candidate for gene-specific therapy for HPV-related cervical cancer.
Since its discovery in 1969, EV71 has been recognized as a frequent cause of epidemics of hand-foot-mouse disease (HFMD) associated with several neurological sequelae in a small proportion of cases. In 1998, the largest EV71 outbreak in Taiwan, more than 90000 children with HFMD had been reported. Internal ribosomal entry site (IRES), structured RNA sequence, was thought that it is essential to recruit and activate translation machinery. We focus on prevention of EV71 infection by using small interfering RNA to inhibit IRES of EV71. we have screened effective siRNA against the expression of Taiwan/4643 EV71 IRES expression cassettes by screening system. We attempt to use siRNA we selected to knockdown expression of EV71 IRES. Then, the cell, EV71 infection, would be protected from death, and decreased the activation of EV71.

Adam, E., Berkova, Z., Daxnerova, Z., Icenogle, J., Reeves, W.C., and Kaufman, R.H. (2000). Papillomavirus detection: demographic and behavioral characteristics influencing the identification of cervical disease. Am J Obstet Gynecol 182, 257-264.
Alberi, L., Sgado, P., and Simon, H.H. (2004). Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons. Development 131, 3229-3236.
Anderson, J., Banerjea, A., Planelles, V., and Akkina, R. (2003). Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA. AIDS Res Hum Retroviruses 19, 699-706.
Apt, D., Watts, R.M., Suske, G., and Bernard, H.U. (1996). High Sp1/Sp3 ratios in epithelial cells during epithelial differentiation and cellular transformation correlate with the activation of the HPV-16 promoter. Virology 224, 281-291.
Baker, T.S., Newcomb, W.W., Olson, N.H., Cowsert, L.M., Olson, C., and Brown, J.C. (1991). Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys J 60, 1445-1456.
Belinson, J., Qiao, Y.L., Pretorius, R., Zhang, W.H., Elson, P., Li, L., Pan, Q.J., Fischer, C., Lorincz, A., and Zahniser, D. (2001). Shanxi Province Cervical Cancer Screening Study: a cross-sectional comparative trial of multiple techniques to detect cervical neoplasia. Gynecol Oncol 83, 439-444.
Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366.
Bonnal, S., Boutonnet, C., Prado-Lourenco, L., and Vagner, S. (2003). IRESdb: the Internal Ribosome Entry Site database. Nucleic Acids Res 31, 427-428.
Braasch, D.A., Paroo, Z., Constantinescu, A., Ren, G., Oz, O.K., Mason, R.P., and Corey, D.R. (2004). Biodistribution of phosphodiester and phosphorothioate siRNA. Bioorg Med Chem Lett 14, 1139-1143.
Brown, B.A., and Pallansch, M.A. (1995). Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus. Virus Res 39, 195-205.
Brummelkamp, T.R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.
Burd, E.M. (2003). Human papillomavirus and cervical cancer. Clin Microbiol Rev 16, 1-17.
Clarke, A.R., Purdie, C.A., Harrison, D.J., Morris, R.G., Bird, C.C., Hooper, M.L., and Wyllie, A.H. (1993). Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362, 849-852.
Doorbar, J. (2006). Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond) 110, 525-541.
Dyson, N., Howley, P.M., Munger, K., and Harlow, E. (1989). The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934-937.
Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 20, 6877-6888.
Favre, M. (1975). Structural polypeptides of rabbit, bovine, and human papillomaviruses. J Virol 15, 1239-1247.
Filman, D.J., Syed, R., Chow, M., Macadam, A.J., Minor, P.D., and Hogle, J.M. (1989). Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus. Embo J 8, 1567-1579.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.
Ge, Q., McManus, M.T., Nguyen, T., Shen, C.H., Sharp, P.A., Eisen, H.N., and Chen, J. (2003). RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci U S A 100, 2718-2723.
Giladi, H., Ketzinel-Gilad, M., Rivkin, L., Felig, Y., Nussbaum, O., and Galun, E. (2003). Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 8, 769-776.
Greve, J.M., Davis, G., Meyer, A.M., Forte, C.P., Yost, S.C., Marlor, C.W., Kamarck, M.E., and McClelland, A. (1989). The major human rhinovirus receptor is ICAM-1. Cell 56, 839-847.
Guo, S., and Kemphues, K.J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611-620.
Hamilton, W.A., Butler, P.D., Magid, L.J., Han, Z., and Slawecki, T.M. (1999). Fast relaxation of a hexagonal Poiseuille shear-induced near-surface phase in a threadlike micellar solution. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics 60, R1146-1149.
Hammond, S.M., Bernstein, E., Beach, D., and Hannon, G.J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293-296.
Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R., and Hannon, G.J. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146-1150.
Hellen, C.U., and Sarnow, P. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 15, 1593-1612.
Hellen, C.U., and Wimmer, E. (1995). Translation of encephalomyocarditis virus RNA by internal ribosomal entry. Curr Top Microbiol Immunol 203, 31-63.
Huang, F., Khvorova, A., Marshall, W., and Sorkin, A. (2004). Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J Biol Chem 279, 16657-16661.
Hung, C.F., Lu, K.C., Cheng, T.L., Wu, R.H., Huang, L.Y., Teng, C.F., and Chang, W.T. (2006). A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells. Biochem Biophys Res Commun 346, 707-720.
Iizuka, N., Yonekawa, H., and Nomoto, A. (1991). Nucleotide sequences important for translation initiation of enterovirus RNA. J Virol 65, 4867-4873.
Jackson, R.J., Howell, M.T., and Kaminski, A. (1990). The novel mechanism of initiation of picornavirus RNA translation. Trends Biochem Sci 15, 477-483.
Jang, S.K. (2006). Internal initiation: IRES elements of picornaviruses and hepatitis c virus. Virus Res 119, 2-15.
Jang, S.K., and Wimmer, E. (1990). Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev 4, 1560-1572.
Johnson, D.G., Schwarz, J.K., Cress, W.D., and Nevins, J.R. (1993). Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365, 349-352.
Kartasheva, N.N., Contente, A., Lenz-Stoppler, C., Roth, J., and Dobbelstein, M. (2002). p53 induces the expression of its antagonist p73 Delta N, establishing an autoregulatory feedback loop. Oncogene 21, 4715-4727.
Ketting, R.F., Haverkamp, T.H., van Luenen, H.G., and Plasterk, R.H. (1999). Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133-141.
Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M.J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20, 500-505.
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S., et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415-419.
Lin, T.Y., Twu, S.J., Ho, M.S., Chang, L.Y., and Lee, C.Y. (2003). Enterovirus 71 outbreaks, Taiwan: occurrence and recognition. Emerg Infect Dis 9, 291-293.
Lipardi, C., Wei, Q., and Paterson, B.M. (2001). RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297-307.
Lowe, S.W., Schmitt, E.M., Smith, S.W., Osborne, B.A., and Jacks, T. (1993). p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, 847-849.
Lowy, D.R., and Schiller, J.T. (2006). Prophylactic human papillomavirus vaccines. J Clin Invest 116, 1167-1173.
Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563-574.
McCright, I.J., Tsunoda, I., Whitby, F.G., and Fujinami, R.S. (1999). Theiler's viruses with mutations in loop I of VP1 lead to altered tropism and pathogenesis. J Virol 73, 2814-2824.
McMinn, P.C. (2002). An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev 26, 91-107.
Mendelsohn, C., Johnson, B., Lionetti, K.A., Nobis, P., Wimmer, E., and Racaniello, V.R. (1986). Transformation of a human poliovirus receptor gene into mouse cells. Proc Natl Acad Sci U S A 83, 7845-7849.
Mendelsohn, C.L., Wimmer, E., and Racaniello, V.R. (1989). Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56, 855-865.
Miyagishi, M., and Taira, K. (2002). U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 20, 497-500.
Napoli, C., Lemieux, C., and Jorgensen, R. (1990). Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289.
Nicholson, R., Pelletier, J., Le, S.Y., and Sonenberg, N. (1991). Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies. J Virol 65, 5886-5894.
Nomoto, A., Kitamura, N., Golini, F., and Wimmer, E. (1977). The 5'-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg. Proc Natl Acad Sci U S A 74, 5345-5349.
Novina, C.D., Murray, M.F., Dykxhoorn, D.M., Beresford, P.J., Riess, J., Lee, S.K., Collman, R.G., Lieberman, J., Shankar, P., and Sharp, P.A. (2002). siRNA-directed inhibition of HIV-1 infection. Nat Med 8, 681-686.
Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., and Conklin, D.S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16, 948-958.
Paddison, P.J., Silva, J.M., Conklin, D.S., Schlabach, M., Li, M., Aruleba, S., Balija, V., O'Shaughnessy, A., Gnoj, L., Scobie, K., et al. (2004). A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427-431.
Pan, H., and Griep, A.E. (1994). Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development. Genes Dev 8, 1285-1299.
Paul, C.P., Good, P.D., Winer, I., and Engelke, D.R. (2002). Effective expression of small interfering RNA in human cells. Nat Biotechnol 20, 505-508.
Pilipenko, E.V., Gmyl, A.P., Maslova, S.V., Svitkin, Y.V., Sinyakov, A.N., and Agol, V.I. (1992). Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA. Cell 68, 119-131.
Reinhart, B.J., Slack, F.J., Basson, M., Pasquinelli, A.E., Bettinger, J.C., Rougvie, A.E., Horvitz, H.R., and Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906.
Roden, R.B., Hubbert, N.L., Kirnbauer, R., Christensen, N.D., Lowy, D.R., and Schiller, J.T. (1996). Assessment of the serological relatedness of genital human papillomaviruses by hemagglutination inhibition. J Virol 70, 3298-3301.
Romano, N., and Macino, G. (1992). Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6, 3343-3353.
Salo, R.J., and Cliver, D.O. (1976). Effect of acid pH, salts, and temperature on the infectivity and physical integrity of enteroviruses. Arch Virol 52, 269-282.
Sapp, M., Volpers, C., Muller, M., and Streeck, R.E. (1995). Organization of the major and minor capsid proteins in human papillomavirus type 33 virus-like particles. J Gen Virol 76 ( Pt 9), 2407-2412.
Schwarz, D.S., Hutvagner, G., Haley, B., and Zamore, P.D. (2002). Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell 10, 537-548.
Semizarov, D., Kroeger, P., and Fesik, S. (2004). siRNA-mediated gene silencing: a global genome view. Nucleic Acids Res 32, 3836-3845.
Shuey, D.J., McCallus, D.E., and Giordano, T. (2002). RNAi: gene-silencing in therapeutic intervention. Drug Discov Today 7, 1040-1046.
Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H., and Schreiber, R.D. (1998). How cells respond to interferons. Annu Rev Biochem 67, 227-264.
Storey, A., Thomas, M., Kalita, A., Harwood, C., Gardiol, D., Mantovani, F., Breuer, J., Leigh, I.M., Matlashewski, G., and Banks, L. (1998). Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 393, 229-234.
Sun, L., Deng, L., Ea, C.K., Xia, Z.P., and Chen, Z.J. (2004). The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell 14, 289-301.
Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., and Mello, C.C. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123-132.
Thompson, S.R., and Sarnow, P. (2003). Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved. Virology 315, 259-266.
Tijsterman, M., Ketting, R.F., and Plasterk, R.H. (2002). The genetics of RNA silencing. Annu Rev Genet 36, 489-519.
Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P., and Sharp, P.A. (1999). Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13, 3191-3197.
Werness, B.A., Levine, A.J., and Howley, P.M. (1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76-79.
White, E. (1994). Tumour biology. p53, guardian of Rb. Nature 371, 21-22.
Wien, M.W., Curry, S., Filman, D.J., and Hogle, J.M. (1997). Structural studies of poliovirus mutants that overcome receptor defects. Nat Struct Biol 4, 666-674.
Wimmer, E., Hellen, C.U., and Cao, X. (1993). Genetics of poliovirus. Annu Rev Genet 27, 353-436.
Wu, X., and Levine, A.J. (1994). p53 and E2F-1 cooperate to mediate apoptosis. Proc Natl Acad Sci U S A 91, 3602-3606.
Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33.
Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., and Schultz, P.G. (2004). An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc Natl Acad Sci U S A 101, 135-140.
zur Hausen, H. (1996). Viruses in human tumors--reminiscences and perspectives. Adv Cancer Res 68, 1-22.
zur Hausen, H. (1998). Papillomavirus and p53. Nature 393, 217.
zur Hausen, H. (1999). Papillomaviruses in human cancers. Proc Assoc Am Physicians 111, 581-587.