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研究生:高雅君
研究生(外文):Ya Jun Kao
論文名稱:EBVmiRNAebv-miR-BART1-5p之功能性探討及其標的基因的找尋
論文名稱(外文):Functional characterization and target exploration of
指導教授:張玉生張玉生引用關係
指導教授(外文):Y. S. Chang
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:103
中文關鍵詞:人類皰疹病毒4
外文關鍵詞:EBV miRNA ebv-miR-BART1-5p
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microRNAs (miRNAs)是一種長度大約19~25個核苷酸的小RNA分子,可以藉由結合在target mRNA的3’UTR而調控gene的表現。許多文獻指出miRNAs與細胞的分化、生長,以及癌症的形成都有關係。EBV與亞洲人所好發的鼻咽癌 (nasopharyngeal carcinoma,NPC) 關係密切,但是EBV在癌症形成過程中扮演什麼樣的角色目前尚未明瞭。近來發現NPC癌症組織中有EBV miRNAs存在,這些miRNAs可能促進癌症的形成,也可能幫助病毒生長。目前總共有39個EBV mature miRNAs被發現,當中有35個是源自BARTs (BamHI-A rightward transcripts) 片段,其中包括ebv-miR-BART1-5p在內的BART-cluster-I miRNAs可以調控病毒蛋白LMP1的表現,但是這些miRNAs是否會進一步影響宿主細胞的基因表現目前還是未知數。

為了探討ebv-miR-BART1-5p對宿主細胞的影響,是否促進NPC癌症的形成,或是參予免疫調控路徑。我們一方面將ebv-miR-BART1-5的expressing vector送入細胞中,以Q-PCR技術確定expressing vector的表現,一方面利用生物資訊分析方法預測ebv-miR-BART1-5在人類細胞中可能的target gene,並且以Q-PCR技術偵測所預測之target gene mRNA表現,從結果可見HOXA11, TNIP2, E2F4, STAT2, BRF1, C1QTNF8, CHMP6, SH2B1等預測之target gene的mRNA表現可能受到ebv-miR-BART1-5的抑制。再利用Reporter assay偵測這些預測的target gene在protein表現層次上是否受ebv-miR-BART1-5影響,從結果我們發現BRF1, HOXA11, SH2B1等預測之target gene有可能受ebv-miR-BART1-5影響而使其protein表現量降低。由於實驗所得之表現抑制量稍低,因此需再調整reporter assay之實驗條件以確定BRF1, HOXA11, SH2B1等是否真為ebv-miR-BART1-5p之target gene。
Epstein–Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), but the role of EBV in the NPC tumorigenesis remains to be clarified. EBV was the first human virus found to encode microRNAs (miRNAs). A total of 39 mature ebv miRNAs have been identified. In NPC, EBV highly expressed BamHI-A rightward transcripts (BARTs). BARTs region could generate 35 miRNAs, some of these BART miRNAs, including miR-BART1-5p, could negatively regulate viral protein LMP1. But the function of most BART miRNAs is unknown. This study aimed to investigate the effect of ebv-miR-BART1-5p on host cellular proteins and its role in NPC tumorigenesis.

To study the role of miR-BART1-5p, we constructed the expressing vector of miR-BART1-5p, and used highly sensitive stem-loop RT-PCR method to detect the expression level of miRNA in cell lines over expressing miR-BART1-5p. On the other hand, we used bioinformatics method to predict potential target genes of miR-BART1-5p. We used RT-PCR to detect the expression level of target mRNA. We found mRNA levels of BRF1, HOXA11 and SH2B1 were reduced in cells overexpressing ebv-miR-BART1-5p. Reporter assay confirmed the interaction between ebv-miR-BART1-5p and the ebv-miR-BART1-5p binding sites on the 3’UTR of BRF1, HOXA11 and SH2B1. These results indicate that BRF1, HOXA11 and SH2B1 are potential targets of ebv-miR-BART1-5p.
論文指導教授推薦書………………………………………………..….i
論文口試委員審定書…………………………………………………..ii
長庚大學授權書…………………………………………………….…iii
誌謝…………………………………………………………………….iv
目錄…………………………………………………………………......v
中文摘要…………………………………………………………….....vi
英文摘要……………………………………………………...............viii
前言…………………………………………………………………….1
研究目的……………………………………………………………….8
材料與方法……………………………………………………………10
實驗結果………………………………………………………………22
討論……………………………………………………………………34
實驗結果圖表………………………………………………………….40
參考文獻……………………………………………………………….78
附錄…………………………………………………………………….82
van Beek, J., Brink, A.A., Vervoort, M.B., van Zijp, M.J., Meijer, C.J., van den Brule, A.J., and Middeldorp, J.M. (2003). In vivo transcription of the Epstein-Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders. The Journal of general virology 84, 2647-2659.

Barth, S., Pfuhl, T., Mamiani, A., Ehses, C., Roemer, K., Kremmer, E., Jaker, C.,
Brennecke, J., Stark, A., Russell, R.B., and Cohen, S.M. (2005). Principles of microRNA-target recognition. PLoS biology 3, e85.

Bushati, N., and Cohen, S.M. (2007). microRNA functions. Annual review of cell and developmental biology 23, 175-205.

Barth, S., Pfuhl, T., Mamiani, A., Ehses, C., Roemer, K., Kremmer, E., Jaker, C., Hock, J., Meister, G., and Grasser, F.A. (2008). Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. Nucleic acids research 36, 666-675.

Cai, X., Schafer, A., Lu, S., Bilello, J.P., Desrosiers, R.C., Edwards, R., Raab-Traub, N., and Cullen, B.R. (2006). Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS pathogens 2, e23.

Cameron, J.E., Yin, Q., Fewell, C., Lacey, M., McBride, J., Wang, X., Lin, Z., Schaefer, B.C., and Flemington, E.K. (2008). Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways. Journal of virology 82, 1946-1958.

Chen, C., Ridzon, D.A., Broomer, A.J., Zhou, Z., Lee, D.H., Nguyen, J.T., Barbisin, M., Xu, N.L., Mahuvakar, V.R., Andersen, M.R., et al. (2005). Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic acids research 33, e179.

Chen, H., Smith, P., Ambinder, R.F., and Hayward, S.D. (1999). Expression of Epstein-Barr virus BamHI-A rightward transcripts in latently infected B cells from peripheral blood. Blood 93, 3026-3032.

Chen, H.L., Lung, M.M., Sham, J.S., Choy, D.T., Griffin, B.E., and Ng, M.H. (1992). Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells. Virology 191, 193-201.

Doench, J.G., and Sharp, P.A. (2004). Specificity of microRNA target selection in translational repression. Genes & development 18, 504-511.

Du, T., and Zamore, P.D. (2005). microPrimer: the biogenesis and function of microRNA. Development (Cambridge, England) 132, 4645-4652.

Esquela-Kerscher, A., and Slack, F.J. (2006). Oncomirs - microRNAs with a role in cancer. Nature reviews 6, 259-269.

Fukayama, M., Hino, R., and Uozaki, H. (2008). Epstein-Barr virus and gastric carcinoma: virus-host interactions leading to carcinoma. Cancer science.

Grey, F., Antoniewicz, A., Allen, E., Saugstad, J., McShea, A., Carrington, J.C., and Nelson, J. (2005). Identification and characterization of human cytomegalovirus-encoded microRNAs. Journal of virology 79, 12095-12099.

Grimson, A., Farh, K.K., Johnston, W.K., Garrett-Engele, P., Lim, L.P., and Bartel, D.P. (2007). MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Molecular cell 27, 91-105.

Grundhoff, A., Sullivan, C.S., and Ganem, D. (2006). A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses. RNA (New York, NY 12, 733-750.

Gleave, M.E., and Monia, B.P. (2005). Antisense therapy for cancer. Nature reviews 5, 468-479.

Hsu, P.W., Lin, L.Z., Hsu, S.D., Hsu, J.B., and Huang, H.D. (2007). ViTa: prediction of host microRNAs targets on viruses. Nucleic acids research 35, D381-385.

Kim do, N., Chae, H.S., Oh, S.T., Kang, J.H., Park, C.H., Park, W.S., Takada, K., Lee, J.M., Lee, W.K., and Lee, S.K. (2007). Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma. Journal of virology 81, 1033-1036.

Izquierdo, M. (2005). Short interfering RNAs as a tool for cancer gene therapy. Cancer gene therapy 12, 217-227.

Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S., Rice, A., Kamphorst, A.O., Landthaler, M., et al. (2007). A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401-1414.

Lee, Y., Jeon, K., Lee, J.T., Kim, S., and Kim, V.N. (2002). MicroRNA maturation: stepwise processing and subcellular localization. The EMBO journal 21, 4663-4670.

Lewis, B.P., Burge, C.B., and Bartel, D.P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15-20.

Lo, A.K., To, K.F., Lo, K.W., Lung, R.W., Hui, J.W., Liao, G., and Hayward, S.D. (2007). Modulation of LMP1 protein expression by EBV-encoded microRNAs. Proceedings of the National Academy of Sciences of the United States of America 104, 16164-16169.

Murphy, E., Vanicek, J., Robins, H., Shenk, T., and Levine, A.J. (2008). Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs: implications for latency. Proceedings of the National Academy of Sciences of the United States of America 105, 5453-5458.

Nielsen, C.B., Shomron, N., Sandberg, R., Hornstein, E., Kitzman, J., and Burge, C.B. (2007). Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. RNA (New York, NY 13, 1894-1910.

Peters, L., and Meister, G. (2007). Argonaute proteins: mediators of RNA silencing. Molecular cell 26, 611-623.

Pfeffer, S., and Voinnet, O. (2006). Viruses, microRNAs and cancer. Oncogene 25, 6211-6219.

Pfeffer, S., Zavolan, M., Grasser, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D., Sander, C., et al. (2004). Identification of virus-encoded microRNAs. Science (New York, NY 304, 734-736.

Raymond, C.K., Roberts, B.S., Garrett-Engele, P., Lim, L.P., and Johnson, J.M. (2005). Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA (New York, NY 11, 1737-1744.

Rodriguez, A., Griffiths-Jones, S., Ashurst, J.L., and Bradley, A. (2004). Identification of mammalian microRNA host genes and transcription units. Genome research 14, 1902-1910.

Stern-Ginossar, N., Elefant, N., Zimmermann, A., Wolf, D.G., Saleh, N., Biton, M., Horwitz, E., Prokocimer, Z., Prichard, M., Hahn, G., et al. (2007). Host immune system gene targeting by a viral miRNA. Science (New York, NY 317, 376-381.

Sullivan, C.S., Grundhoff, A.T., Tevethia, S., Pipas, J.M., and Ganem, D. (2005). SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435, 682-686.

Umbach, J.L., Kramer, M.F., Jurak, I., Karnowski, H.W., Coen, D.M., and Cullen, B.R. (2008). MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature.

Xia, T., O'Hara, A., Araujo, I., Barreto, J., Carvalho, E., Sapucaia, J.B., Ramos, J.C., Luz, E., Pedroso, C., Manrique, M., et al. (2008). EBV microRNAs in primary lymphomas and targeting of CXCL-11 by ebv-mir-BHRF1-3. Cancer research 68, 1436-1442.

Xing, L., and Kieff, E. (2007). Epstein-Barr virus BHRF1 micro- and stable RNAs during latency III and after induction of replication. Journal of virology 81, 9967-9975.

Yin, Q., McBride, J., Fewell, C., Lacey, M., Wang, X., Lin, Z., Cameron, J., and Flemington, E.K. (2008). MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways. Journal of virology 82, 5295-5306.
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