(3.236.118.225) 您好!臺灣時間:2021/05/14 12:28
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:黃詩茜
研究生(外文):Shih Cian Huang
論文名稱:利用系統生物學方法分析ebv-miR-BART18-5p的功能
論文名稱(外文):Functional analysis of ebv-miR-BART18-5p by systems biology approaches
指導教授:周成功陳華鍵
指導教授(外文):C. K. ChouH. C. Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:59
中文關鍵詞:愛潑斯坦-巴爾二氏病毒微小核糖核酸系統生物學
外文關鍵詞:EBVmicroRNAsystems biology
相關次數:
  • 被引用被引用:0
  • 點閱點閱:129
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
愛潑斯坦-巴爾二氏病毒 (Epstein-Barr Virus; EBV) 感染後的潛伏期被認為可能與某些癌症的發生有關,其中包含好發於東南亞地區的鼻咽癌。研究已知,某些由 EBV 所編碼的 microRNAs (miRNAs) 可調節病毒的基因表現,或是影響宿主的免疫反應或細胞存活能力。但大部分 EBV miRNAs 的功能至今尚未清楚。我們先前已利用反轉錄聚合酶連鎖反應檢測鼻咽癌組織檢體中 EBV miRNAs 的表現量,發現 ebv-miR-BART18-5p (BART18-5p) 的表現量在所有 EBV 的 miRNA 中最高。有鑑於此,我們利用電腦預測及生物晶片方法,配合系統生物學分析,來研究 BART18-5p 的作用途徑。
利用生物晶片分析穩定表現 BART18-5p 的細胞株及控制組,以 p-value 小於 0.05,差異倍數大於 1.1 倍,篩選出 1018 個基因,在 MetaCore 5.1 分析作用途徑,發現細胞外基質重組途徑中,Matrix metallopeptidase 9 (MMP9) 基因在過度表現 BART18-5p 組中的表現量明顯較高。利用 Q-PCR 與西方點墨法,證明過度表現 BART18-5p 會增加 MMP9 基因與蛋白質的表現。進一步利用報導基因檢測 MMP9 上游轉錄活化子活性,發現過度表現 BART18-5p 會使 MMP9 轉錄活化子 NF-κB 及 AP-1 的活性增加。最後,尋找 BART18-5p 調控 NF-κB 及 AP-1 活性的標的基因,以報導基因證明 BART18-5p 會抑制 NF-κB 及 AP-1 的抑制劑 RBPJ 與 PIAS1。綜合以上所述,我們認為,BART18-5p 可能藉由抑制 RBPJ 和 PIAS1,使 NF-kB 和 AP-1 的活性增加,進而促進 MMP9 的表現。這些作用最終可能會影響鼻咽癌細胞的增生、存活、侵襲能力及免疫反應等。
Latent Epstein-Barr virus infection is associated with several cancers, including nasopharyngeal carcinoma (NPC) that is especially common in Southern China. Recently, some of the EBV-encode miRNAs have been found to play important roles in regulating EBV gene expression or host immune responses and survival. But the functions of most EBV miRNAs remain largely unknown. Using stem-loop RT-PCR to detect the expression level of EBV miRNAs in NPC tissues, we found that the expression level of ebv-miR-BART18-5p was higher than other EBV miRNAs. We using computational prediction, microarray and systems biology approaches to investigate the functional pathways that are regulated by BART18-5p.
We compared the mRNA levels in control with BART18-5p over-expressing stable cell lines by microarray, and generated a differentially expressed gene list with 1018 genes (p-value < 0.05, fold change > 1.1). By analyzing the differentially expressed genes in MetaCore 5.1, we found that the expression level of MMP9 in ECM remodeling pathway is significantly elevated. Using Q-PCR and western blot, we demonstrated that BART18-5p could induce MMP9 gene expression in mRNA and protein levels. We further analyzed the activities of transcription activator of MMP9 by promoter reporter assay. The results showed that the activities of NF-kB and AP-1 were up-regulated by BART18-5p. Furthermore, by using pMIR-luciferase reporter assay, we demonstrated that the inhibitors of NF-kB and AP-1, such as RBPJ and PIAS1, could be inhibited by BART18-5p. Collectively, our findings suggest that BRAT18-5p could inhibit RBPJ and PIAS1 to activate NF-kB and AP-1, and sequentially up-regulate MMP9 expression. These events may lead to regulate host cell proliferation, survival, invasion and immune response in NPC.
目 錄
論文指導教授推薦薦..................................................................................I
論文口試委員審定書................................................................................II
國家圖書館授權書及長庚大學博碩士紙本論文著作授權書...............III
誌謝..........................................................................................................IV
中文摘要....................................................................................................V
英文摘要..................................................................................................VI
目錄.........................................................................................................VII

第一章 緒論
1.1愛潑斯坦-巴爾二氏病毒 (Epstein-Barr virus; EBV)………........1
愛潑斯坦-巴爾二氏病毒………………………………………..1
愛潑斯坦-巴爾二氏病毒與疾病………………………………..2
愛潑斯坦-巴爾二氏病毒所編碼的 microRNA……...…………2
1.2 microRNA………………………………………………………....6
microRNA……………………………………………..…………6
microRNA 的生成與作用…………………………...………….6
研究 microRNA 作用標的及功能的方法………..……………8
1.3 研究動機與目的………………………………………………..10
1.4 實驗流程……………………………………….………………..11

第二章 材料與方法
2.1 細胞株………………………………………..…………….……12
2.2 EBV miRNA 的選殖…………………...………………….…..12
2.3 穩定性轉染 (stable transfection)……………...……………......12
2.4 RNA 抽取…………………………………..…………….……14
2.5 反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase
chain reaction, RT-PCR)………………………..………….…....14
2.6 同步即時定量聚合酶連鎖反應 ( Quantitative real-time
polymerase chain reaction, Q-PCR)………………..…….….….15
2.7 生物晶片分析……………………………………..………….....16
2.8 電腦預測互補序列…………………………………..……….....16
2.9 鼻咽癌檢體生物晶片分析………………………………….......17
2.10 系統生物學分析……………………………………………….17
2.11 西方點墨法…………………………………………………….17
2.12 過渡性轉染 (transient transfection)………………………......18

第三章 結果與討論
3.1 建立穩定表現 ebv-miR-BART18-5p 的細胞株………….......20
3.2 以生物晶片分析穩定表現 ebv-miR-BART18-5p 的 HK1
細胞株..........................................................................................20
3.3 以MetaCore分析BART18-5p的作用途徑…………………….21
3.4 穩定性表現 BART18-5P 細胞株中 MMP9 的表現量............22
3.5 以 MetaCore 進行 Network Analysis………………...……….23
3.6 以報導基因檢測在過度表現 BART18-5p 後,MMP9
上游轉錄活化因子的活性……………………………………..24
3.7 以電腦預測 BART18-5p 的可能標的基因……………….......24
3.8 以 Q-PCR 及報導基因驗證 BART18-5p 的可能標的基因...25
3.9 結論……………………………………………………………...26

參考文獻……………………………………………………...………..28
圖表附錄
【圖1】 EBV microRNA前驅物在EBV基因體上的位置………….32
【圖2】利用 Q-PCR 檢測鼻咽癌組織 CN8 和正常組織
CT8 中 EBV microRNAs 的表現……………………….33
【圖3】利用 Q-PCR 檢測鼻咽癌組織和正常組織中
BART18-5p 的表現量…………………………………….33
【圖4】miRNA的生成與作用…………...…………………….……34
【圖5】實驗流程圖…………………………………………….…....35
【圖6】RT-PCR、Q-PCR 所使用之引子以及其反應步驟….…….36
【圖7】將 BART18-5p 穩定性轉染至細胞株中,並以
Q-PCR 觀察 BART18-5p 表現量……………………….37
【圖8】生物晶片分析.........................................................................38
【圖9】BART18-5p array 或 Random gene set 在 MetaCore
比對GeneGo Pathway Maps 所得的作用途徑..................38
【圖10】Cell adhesion_ECM remodeling 作用途徑........................ 39

【圖11】穩定表現 BART18-5p 及鼻咽癌檢體生物晶片中
MMPs 表現量的 PCA 示意圖…………………...…..40
【圖12】穩定表現 BART18-5P 之細胞株中 MMP9 基因
的表現量…………………………………………………41
【圖13】穩定表現 BART18-5P 之細胞株中 MMP9 蛋白
的表現量…………………………………………………42
【圖14】MMP9 的轉錄活化因子與其下游所調控的基因…….....43
【圖15】以報導基因檢測 BART18-5p 對 MMP9 上游轉
錄活化因子活性的影響…………………………………44
【圖16】以Q-PCR檢測CBP, NF-kB, c-Fos上游抑制因子的
表現量……………………………………………………45
【圖17】利用冷光酶報導基因檢測 BART18-5p 的可能標
的基因……………………………………………………46
【圖18】利用 LNA 與冷光酶報導基因驗證 BART18-5p
的標的…………………………………………………..47
【圖19】BART18-5p 可能的作用途徑.............................................48
【圖20】EBV 調控宿主細胞 NF-κB、AP-1 與 MMP9 的途徑..48
【表1】EBV潛伏期所表現的病毒基因與相關疾病……………....32
【表2】MMP 基因表現的差異倍數與 P 值……………….......…40
【表3】BART18-5p array Network list…………………………..,...42
【表4】CBP, NF-kB, c-Fos上游的抑制因子…………………….....45
1. Epstein MA, Achong BG, Barr YM (1964) Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 1: 702-703.
2. Henle W, Diehl V, Kohn G, Zur Hausen H, Henle G (1967) Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157:1064-1065.
3.Nemerow, G.R., Moore, M.D., Cooper, N.R. (1990) Structure and function of the B-lymphocyte Epstein-Barr virus/C3d receptor. Adv. Cancer Res 54: 273-300.
4. Kikuta, H., Taguchi, Y., Tomizawa, K., Kojima, K., Kawamura, N., et al. (1988) Epstein-Barr virus genome-positive T lymphocytes in a boy with chronic active EBV infection associated with Kawasaki-like disease. Nature 333: 455-457.
5. Sixbey, J. W., Vesterinen, E. H., Nedrud, J. G., Raab-Traub, N., Walton, L. A., et al. (1983) Replication of Epstein-Barr virus in human epithelial cells infected in vitro. Nature 306:480-483.
6. Thompson MP, Kurzrock R (2004) Epstein-Barr virus and cancer. Clin Cancer Res. 10 :803-821.
7. Yamamoto N, Takizawa T, Iwanaga Y, Shimizu N, Yamamoto N (2000) Malignant transformation of B lymphoma cell line BJAB by Epstein-Barr virus-encoded small RNAs. FEBS Lett 484:153-8.
8. Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., et al. (1984) DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310:207-211.
9. Hitt MM, Allday MJ, Hara T, Karran L, Jones MD., et al. (1989) EBV gene expression in an NPC-related tumour. EMBO J 8 :2639-2651.
10. Pfeffer S, Zavolan M, Grässer FA, Chien M, Russo JJ,, et al. (2004) Identification of virus-encoded microRNAs. Science 304:734-736.
11. Grundhoff A, Sullivan CS, Ganem D (2006) A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses. RNA 12 :733-750.
12. Kim do N, Chae HS, Oh ST, Kang JH, Park CH,et al. (2007) Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma. J Virol 81:1033-1036.
13. Edwards RH, Marquitz AR, Raab-Traub N. (2008) Epstein-Barr Virus BART MicroRNAs Are Produced from a Large Intron prior to Splicing. J Virol 82:9094-106.
14. Chen H, Huang J, Wu FY, Liao G, Hutt-Fletcher L,et al. (2005) Regulation of expression of the Epstein-Barr virus BamHI-A rightward transcripts. J Virol 79:1724-1733.
15. Barth S, Pfuhl T, Mamiani A, Ehses C, Roemer K, et al. (2008) Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. Nucleic Acids Res 36:666-675.
16. Lo AK, To KF, Lo KW, Lung RW, Hui JW, et al. (2007) Modulation of LMP1 protein expression by EBV-encoded microRNAs. Proc Natl Acad Sci U S A 104:16164-9.
17. Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, et al. (2008) An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival. J Exp Med 205:2551-60.
18. Nachmani D, Stern-Ginossar N, Sarid R, Mandelboim O. (2009) Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells. Cell Host Microbe 5:376-85.
19. Xia T, O'Hara A, Araujo I, Barreto J, Carvalho E, et al. (2008) EBV microRNAs in primary lymphomas and targeting of CXCL-11 by ebv-miR-BHRF1-3. Cancer Res 68 :1436-1442.
20. Napoli C, Lemieux C, 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.
21. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843-854.
22. Kim VN (2005) Small RNAs: classification, biogenesis, and function. Mol Cells 19:1-15.
23. He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522-531.
24. Rana TM (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8:23-36.
25. Winter J, Jung S, Keller S, Gregory RI, Diederichs S. (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11:228-34
26. Naqvi AR, Islam MN, Choudhury NR, Haq QM. (2009) The fascinating world of RNA interference. Int J Biol Sci 5:97-117.
27. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433:769-773.
28. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, et al. (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 27:91-105.
29. Bartel DP. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215-33.
30. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102-114.
31. Chu CY, Rana TM (2006) Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol 4:e210.
32. Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W (2006) Relief of microRNA-mediated translational repression in human cells subjected to stress.Cell 125:1111-1124.
33. Hendrickson DG, Hogan DJ, Herschlag D, Ferrell JE, Brown PO (2008) Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance. PLoS ONE 3 :e2126.
34. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of mammalian microRNA targets. Cell 115:787-98.
35. Papagiannakopoulos T, Shapiro A, Kosik KS. (2008) MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Res 68:8164-72.
36. Dodd LE, Sengupta S, Chen IH, den Boon JA, Cheng YJ et al. (2008) Genes Involved in DNA Repair and Nitrosamine Metabolism and Those Located on Chromosome 14q32 Are Dysregulated in Nasopharyngeal Carcinoma Cancer. Epidemiol Biomarkers Prev 15:2216-25.
37. Lee DC, Chua DT, Wei WI, Sham JS, Lau AS. (2007) Induction of matrix metalloproteinases by Epstein-Barr virus latent membrane protein 1 isolated from nasopharyngeal carcinoma. Biomed Pharmacother 61:520-6.
38. Egeblad M, Werb Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2:161-74.
39. Lu J, Chua HH, Chen SY, Chen JY, Tsai CH. (2003) Regulation of matrix metalloproteinase-1 by EpsteineBarr virus proteins. Cancer Res 63: 256-62.
40. Yoshizaki T, Sato H, Furukawa M, Pagano JS. (1998) The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1. Proc Natl Acad Sci 95:3621-6.
41. Horikawa T, Yoshizaki T, Sheen TS, Lee SY, Furukawa M. (2003) Association of latent membrane protein 1 and matrix metalloproteinase 9 with metastasis in nasopharyngeal carcinoma. Cancer 89:715-23.
42. Tang JG, Li X, Chen P. (2004) Expression of matrix metalloproteinase 9 in nasopharyngeal carcinoma and association with Epstein-Barr virus infection. J Zhejiang Univ Sci 5:1304-12.
43. Wang C, Deng X, Li X, Gu H, Yi W, et al (2002) Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus LMP1 via NF-kappa B or AP-1 signaling pathway in nasopharyngeal carcinoma cells. Zhonghua Zhong Liu Za Zhi 24:9-13.
44. Yoshizaki T, Sato H, Murono S, Pagano JS, Furukawa M. (1999) Matrix metalloproteinase 9 is induced by the Epstein-Barr virus BZLF1 transactivator. Clin Exp Metastasis 17:431-6.
45. Yip TT, Ngan RK, Lau WH, Poon YF, Joab I, et al (1994) A possible prognostic role of immunoglobulin-G antibody against recombinant Epstein-Barr virus BZLF-1 transactivator protein ZEBRA in patients with nasopharyngeal carcinoma. Cancer 74:2414-24.
46. Tsao SW, Tramoutanis G, Dawson CW, Lo AK, Huang DP. (2002) The significance of LMP1 expression in nasopharyngeal carcinoma. Semin Cancer Biol 12:473-87.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔