(34.204.201.220) 您好!臺灣時間:2021/04/20 11:25
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:黃至韡
研究生(外文):Chih-Wei Huang
論文名稱:日本腦炎病毒持續性感染細胞株大量表現之miR-125b-5p 功能之分析
論文名稱(外文):Characterization of highly expressed miR-125b-5p in Japanese encephalitis virus persistently-infected cells
指導教授:張瑞宜張瑞宜引用關係
指導教授(外文):Ruey-Yi Chang
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
論文頁數:56
中文關鍵詞:日本腦炎病毒持續性感染
外文關鍵詞:Japanese encephalitis viruspersistent infectionmicroRNA
相關次數:
  • 被引用被引用:0
  • 點閱點閱:38
  • 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:4
  • 收藏至我的研究室書目清單書目收藏:0
許多研究證明宿主或病毒產生的microRNAs (miRNAs)在調控基因表現中具有多功能的角色,然而在黃質病毒屬的病毒中,miRNAs如何參與及建立持續性感染的機制仍然是未知。我們先前的研究顯示,日本腦炎病毒感染哺乳動物的細胞(倉鼠腎臟細胞)可以造成持續性感染。為了分析miRNAs 於持續性感染細胞中所扮演的角色,利用小RNA
深度測序技術,比較在急性感染和持續性感染細胞之間miRNAs 表現差異性,結果顯示在持續性感染細胞中miR-125b-5p 會大量的表現。本研究我更進一步利用北方墨點法及定量即時聚合酶鏈鎖反應(qRT-PCR),再次證實miR-125b-5p 會在持續性感染細胞中大量表現。分析由急性感染進入持續性感染的過程中miR-125b-5p 的表現量,證明一當持續性感染產生時,miR-125b-5p 即有顯著性的增加。此外,miR-125b-5p 轉染的劑量會影響細胞存活量,當miR-125b-5p 轉染濃度低於5 nM 時,細胞的存活率會有些微的增加,但在轉染高濃度的miR-125b-5p (25 nM)時,則會降低細胞的存活率。在轉染miR-125b-5p(0.5 nM)的情況下,並不會挽救因病毒感染引起的細胞死亡,但是會明顯降低病毒的力價。利用生物資訊的工具,找出六個miR-125b-5p 可能的目標基因,qRT-PCR 實驗證明這些目標基因在持續性感染細胞中表現量都會減少,並且利用luciferase reporter assay 的方式進一步證明miR-125b-5p 確實會和這些目標基因結合。綜合以上結果顯示,在日本腦炎病毒持續性感染的細胞中miR-125b-5p 會有大量的表現,而且miR-125b-5p 會抑制病毒的複製,推測miR-125b-5p 是經由標靶相關訊息傳遞路徑的調控因子,降低病毒的複製,抑制細胞增生的速率或細胞凋亡的產生,進而使得細胞與病毒達成和平共處而進入持續性感染。本研究是首次報導黃質病毒屬病毒持續性感染的細胞中miRNAs 具有
潛在的調控能力。
A number of studies have been reported that host or virus-derived
microRNAs (miRNAs) have participated versatile roles in multiple
biological processes. How miRNAs may have involved in the establishment of persistent infection with flaviviruses remains mostly unknown. We have shown previously that Japanese encephalitis virus (JEV) could establish persistent infection in mammalian (BHK-21) cells. To characterize the potential roles of miRNAs in the establishment of persistent infection, small RNA deep sequencing approach was performed to obtain the miRNA expression profile in comparison between the acute and persistent infection. The results showed that miR-125b-5p is the most abundant expressed one in JEV persistently infected cells. In this study, I further characterized the abundance of miR-125b-5p by northern blot and quantitative RT-PCR. I demonstrated that as soon as the cells established persistent infection, the significant high expression of miR-125b-5p was readily observed. Transfecting of miR-125b-5p at low dosage (< 5 nM) slightly increased cell
viability, while high concentration at 25 nM significantly reduced cell viability indicting that miR-125b-5p has dose effect. Transfecting of miR-125b-5p at 0.5 nM did not rescue virus-induced cell death but significantly reduced virus titers. Six potential targets of miR-125b-5p were predicted using bioinformatics tools. These targets are down regulated in persistently infected cells and are direct targets of miR-125b-5p as evidenced by luciferase reporter assay. Taken together, these results demonstrated that
miR-125b-5p up regulated in persistently infected cells, inhibited virus replication presumably by targeting to several host regulators in signal transduction pathway that could lead to persistent infection. This is the first report that characterized potential roles of miRNAs in the establishment of flaviviral persistent infection.
Chapter 1. INTRODUCTION ........................................ 1
1. Japanese encephalitis virus.................................. 1
2. JEV genome organization and the sfRNA ....................... 1
3. Noncoding RNAs (ncRNAs) ..................................... 3
4. MicroRNAs ................................................... 4
5. Purpose of this study ....................................... 7
Chapter 2. MATERIALS AND METHODS ............................... 9
1. Cells and viruses ........................................... 9
2. Virus amplification ......................................... 9
3. Plaque assay ............................................... 10
4. Establishment of JEV persistently infected BHK-21 cell line. 10
5. RNA extraction ............................................. 11
6. MicroRNA northern blot analysis ............................ 12
7. Quantitative real-time polymerase chain reaction (qPCR) .... 14
8. MTT cell viability assay ................................... 16
9. Prediction and cloning of miR-125b-5p target sequences ..... 17
10. Luciferase reporter assay ................................. 18
Chapter 3. RESULTS ............................................ 21
1. MiR-125b-5p is highly expressed in persistently infected BHK-21 cells. ........................................................ 21
2. MiR-125b-5p expression increased from acute to persistent infection. .................................................... 22
3. The dosage effect of miR-125b-5p in cell viability. ........ 23
4. Transfecting miR-125b-5p inhibits viral replication......... 25
5. Confirmation of miR-125b-5p targeting genes. ............... 26
Chapter 4. DISCUSSION ......................................... 29
Chapter 5. REFERENCES ......................................... 35
Tables..........................................................41
Figures.........................................................45
Appendix........................................................55
1. Adams, C., G. Cazzanelli, S. Rasul, B. Hitchinson, Y. Hu, R. C. Coombes, S. Raguz, and E. Yague. 2015. Apoptosis inhibitor TRIAP1 is a novel effector of drug resistance. Oncol Rep 34:415-422.
2. Bartel, D. P. 2004. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116:281-297.
3. Borner, C. 2003. The Bcl-2 protein family: sensors and checkpoints for life-or-death decisions. Molecular Immunology 39:615-647.
4. Brinton, M. A., and M. Basu. 2015. Functions of the 3' and 5' genome RNA regions of members of the genus Flavivirus. Virus Res 206:108-119.
5. Bueno, M. J., and M. Malumbres. 2011. MicroRNAs and the cell cycle. Biochim Biophys Acta 1812:592-601.
6. Chang, J., J. T. Guo, D. Jiang, H. Guo, J. M. Taylor, and T. M. Block. 2008. Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells. Journal of virology 82:8215-8223.
7. Chapman, E. G., D. A. Costantino, J. L. Rabe, S. L. Moon, J. Wilusz, J. C. Nix, and J. S. Kieft. 2014. The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production. Science 344:307-310.
8. Chapman, E. G., S. L. Moon, J. Wilusz, and J. S. Kieft. 2014. RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA. Elife 3:e01892.
9. Chen, Z., J. Ye, U. Ashraf, Y. Li, S. Wei, S. Wan, A. Zohaib, Y. Song, H. Chen, and S. Cao. 2016. MicroRNA-33a-5p Modulates Japanese Encephalitis Virus Replication by Targeting Eukaryotic Translation Elongation Factor 1A1. Journal of virology 90:3722-3734.
10. Clarke, B. D., J. A. Roby, A. Slonchak, and A. A. Khromykh. 2015. Functional non-coding RNAs derived from the flavivirus 3' untranslated region. Virus Research 206:53-61.
11. Dolken, L., A. Krmpotic, S. Kothe, L. Tuddenham, M. Tanguy, L. Marcinowski, Z. Ruzsics, N. Elefant, Y. Altuvia, H. Margalit, U. H. Koszinowski, S. Jonjic, and S. Pfeffer. 2010. Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. PLoS Pathog 6:e1001150.
12. Foltz, I. N., R. E. Gerl, J. S. Wieler, M. Luckach, R. A. Salmon, and J. W. Schrader. 1998. Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli. J Biol Chem 273:9344-9351.
13. Funk, A., K. Truong, T. Nagasaki, S. Torres, N. Floden, E. Balmori Melian, J. Edmonds, H. Dong, P. Y. Shi, and A. A. Khromykh. 2010. RNA structures required for production of subgenomic flavivirus RNA. Journal of virology 84:11407-11417.
14. Gottwein, E., and B. R. Cullen. 2008. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe 3:375-387.
15. Hernandez, J. M., D. H. Floyd, K. N. Weilbaecher, P. L. Green, and K. Boris-Lawrie. 2008. Multiple facets of junD gene expression are atypical among AP-1 family members. Oncogene 27:4757-4767.
16. Hussain, M., and S. Asgari. 2014. MicroRNA-like viral small RNA from Dengue virus 2 autoregulates its replication in mosquito cells. Proc Natl Acad Sci U S A 111:2746-2751.
17. Hussain, M., S. Torres, E. Schnettler, A. Funk, A. Grundhoff, G. P. Pijlman, A. A. Khromykh, and S. Asgari. 2012. West Nile virus encodes a microRNA-like small RNA in the 3' untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells. Nucleic Acids Res 40:2210-2223.
18. Ivey, K. N., and D. Srivastava. 2010. MicroRNAs as regulators of differentiation and cell fate decisions. Cell Stem Cell 7:36-41.
19. Le, M. T., N. Shyh-Chang, S. L. Khaw, L. Chin, C. Teh, J. Tay, E. O'Day, V. Korzh, H. Yang, A. Lal, J. Lieberman, H. F. Lodish, and B. Lim. 2011. Conserved regulation of p53 network dosage by microRNA-125b occurs through evolving miRNA-target gene pairs. PLoS Genet 7:e1002242.
20. Le, M. T., C. Teh, N. Shyh-Chang, H. Xie, B. Zhou, V. Korzh, H. F. Lodish, and B. Lim. 2009. MicroRNA-125b is a novel negative regulator of p53. Genes Dev 23:862-876.
21. Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V. N. Kim. 2003. The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415-419.
22. Lima, R. T., S. Busacca, G. M. Almeida, G. Gaudino, D. A. Fennell, and M. H. Vasconcelos. 2011. MicroRNA regulation of core apoptosis pathways in cancer. Eur J Cancer 47:163-174.
23. Lin, K. C., H. L. Chang, and R. Y. Chang. 2004. Accumulation of a 3'-Terminal Genome Fragment in Japanese Encephalitis Virus-Infected Mammalian and Mosquito Cells. Journal of virology 78:5133-5138.
24. Liu, R., L. Yue, X. Li, X. Yu, H. Zhao, Z. Jiang, E. Qin, and C. Qin. 2010. Identification and characterization of small sub-genomic RNAs in dengue 1-4 virus-infected cell cultures and tissues. Biochemical and biophysical research communications 391:1099-1103.
25. Mackenzie, J. S., D. J. Gubler, and L. R. Petersen. 2004. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 10:S98-109.
26. Mattick, J. S., and I. V. Makunin. 2006. Non-coding RNA. Hum Mol Genet 15:R17-29.
27. Mi, J., C. Guo, D. L. Brautigan, and J. M. Larner. 2007. Protein phosphatase-1alpha regulates centrosome splitting through Nek2. Cancer Res 67:1082-1089.
28. Neyts, J., P. Leyssen, and E. De Clercq. 1999. Infections with flaviviridae. Verh K Acad Geneeskd Belg 61:661-697; discussion 697-669.
29. Ninomiya, M., Y. Kondo, O. Kimura, R. Funayama, T. Nagashima, T. Kogure, T. Morosawa, Y. Tanaka, K. Nakayama, and T. Shimosegawa. 2016. The expression of miR-125b-5p is increased in the serum of patients with chronic hepatitis B infection and inhibits the detection of hepatitis B virus surface antigen. J Viral Hepat 23:330-339.
30. Pijlman, G. P., A. Funk, N. Kondratieva, J. Leung, S. Torres, L. van der Aa, W. J. Liu, A. C. Palmenberg, P. Y. Shi, R. A. Hall, and A. A. Khromykh. 2008. A highly structured, nuclease-resistant, noncoding RNA produced by flaviviruses is required for pathogenicity. Cell Host Microbe 4:579-591.
31. Raisch, J., A. Darfeuille-Michaud, and H. T. Nguyen. 2013. Role of microRNAs in the immune system, inflammation and cancer. World J Gastroenterol 19:2985-2996.
32. Roby, J. A., G. P. Pijlman, J. Wilusz, and A. A. Khromykh. 2014. Noncoding subgenomic flavivirus RNA: multiple functions in West Nile virus pathogenesis and modulation of host responses. Viruses 6:404-427.
33. Silva, P. A., C. F. Pereira, T. J. Dalebout, W. J. Spaan, and P. J. Bredenbeek. 2010. An RNA pseudoknot is required for production of yellow fever virus subgenomic RNA by the host nuclease XRN1. Journal of virology 84:11395-11406.
34. Solomon, T., N. M. Dung, R. Kneen, M. Gainsborough, D. W. Vaughn, and V. T. Khanh. 2000. Japanese encephalitis. Journal of Neurology, Neurosurgery & Psychiatry 68:405-415.
35. Sun, Y. M., K. Y. Lin, and Y. Q. Chen. 2013. Diverse functions of miR-125 family in different cell contexts. J Hematol Oncol 6:6.
36. Surdziel, E., M. Cabanski, I. Dallmann, M. Lyszkiewicz, A. Krueger, A. Ganser, M. Scherr, and M. Eder. 2011. Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways. Blood 117:4338-4348.
37. Thounaojam, M. C., D. K. Kaushik, K. Kundu, and A. Basu. 2014. MicroRNA-29b modulates Japanese encephalitis virus-induced microglia activation by targeting tumor necrosis factor alpha-induced protein 3. Journal of neurochemistry 129:143-154.
38. Thounaojam, M. C., K. Kundu, D. K. Kaushik, S. Swaroop, A. Mahadevan, S. K. Shankar, and A. Basu. 2014. MicroRNA 155 regulates Japanese encephalitis virus-induced inflammatory response by targeting Src homology 2-containing inositol phosphatase 1. Journal of virology 88:4798-4810.
39. Tkach, M., C. Rosemblit, M. A. Rivas, C. J. Proietti, M. C. Diaz Flaque, M. F. Mercogliano, W. Beguelin, E. Maronna, P. Guzman, F. G. Gercovich, E. G. Deza, P. V. Elizalde, and R. Schillaci. 2013. p42/p44 MAPK-mediated Stat3Ser727 phosphorylation is required for progestin-induced full activation of Stat3 and breast cancer growth. Endocr Relat Cancer 20:197-212.
40. Tsai, K. N., S. F. Tsang, C. H. Huang, and R. Y. Chang. 2007. Defective interfering RNAs of Japanese encephalitis virus found in mosquito cells and correlation with persistent infection. Virus Res 124:139-150.
41. Tycowski, K. T., Y. E. Guo, N. Lee, W. N. Moss, T. K. Vallery, M. Xie, and J. A. Steitz. 2015. Viral noncoding RNAs: more surprises. Genes Dev 29:567-584.
42. Unni, S. K., D. Ruzek, C. Chhatbar, R. Mishra, M. K. Johri, and S. K. Singh. 2011. Japanese encephalitis virus: from genome to infectome. Microbes Infect 13:312-321.
43. van den Hurk, A. F., S. A. Ritchie, and J. S. Mackenzie. 2009. Ecology and geographical expansion of Japanese encephalitis virus. Annu Rev Entomol 54:17-35.
44. Varkonyi-Gasic, E., R. Wu, M. Wood, E. F. Walton, and R. P. Hellens. 2007. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12.
45. Wang, D., L. Cao, Z. Xu, L. Fang, Y. Zhong, Q. Chen, R. Luo, H. Chen, K. Li, and S. Xiao. 2013. MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-kappaB pathway. PLoS One 8:e55838.
46. Winter, J., S. Jung, S. Keller, R. I. Gregory, and S. Diederichs. 2009. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11:228-234.
47. Wu, L., J. Fan, and J. G. Belasco. 2006. MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A 103:4034-4039.
48. Wu, N., X. Lin, X. Zhao, L. Zheng, L. Xiao, J. Liu, L. Ge, and S. Cao. 2013. MiR-125b acts as an oncogene in glioblastoma cells and inhibits cell apoptosis through p53 and p38MAPK-independent pathways. Br J Cancer 109:2853-2863.
49. Wu, S., F. Liu, L. Xie, Y. Peng, X. Lv, Y. Zhu, Z. Zhang, and X. He. 2015. miR-125b Suppresses Proliferation and Invasion by Targeting MCL1 in Gastric Cancer. Biomed Res Int 2015:365273.
50. Zeng, C. W., X. J. Zhang, K. Y. Lin, H. Ye, S. Y. Feng, H. Zhang, and Y. Q. Chen. 2012. Camptothecin induces apoptosis in cancer cells via microRNA-125b-mediated mitochondrial pathways. Mol Pharmacol 81:578-586.
51. Zhang, B., X. Pan, G. P. Cobb, and T. A. Anderson. 2007. microRNAs as oncogenes and tumor suppressors. Dev Biol 302:1-12.
52. Zhang, L., Y. Ge, and E. Fuchs. 2014. miR-125b can enhance skin tumor initiation and promote malignant progression by repressing differentiation and prolonging cell survival. Genes Dev 28:2532-2546.
53. Zhang, Z., J. Chen, Y. He, X. Zhan, R. Zhao, Y. Huang, H. Xu, Z. Zhu, and Q. Liu. 2014. miR-125b inhibits hepatitis B virus expression in vitro through targeting of the SCNN1A gene. Arch Virol 159:3335-3343.
54. Zhou, M., Z. Liu, Y. Zhao, Y. Ding, H. Liu, Y. Xi, W. Xiong, G. Li, J. Lu, O. Fodstad, A. I. Riker, and M. Tan. 2010. MicroRNA-125b confers the resistance of breast cancer cells to paclitaxel through suppression of pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) expression. J Biol Chem 285:21496-21507.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
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