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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李君男(Chun-Nan Lee) | |
| dc.contributor.author | Bing-Ching Ho | en |
| dc.contributor.author | 何炳慶 | zh_TW |
| dc.date.accessioned | 2021-06-15T07:01:57Z | - |
| dc.date.available | 2013-03-03 | |
| dc.date.copyright | 2011-03-03 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-01-18 | |
| dc.identifier.citation | AbuBakar, S., Chee, H.Y., Al-Kobaisi, M.F., Xiaoshan, J., Chua, K.B., and Lam, S.K. (1999). Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res 61, 1-9.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48557 | - |
| dc.description.abstract | 病毒感染過程中，病毒與宿主間具有複雜的相互作用。病毒感染過程會依賴宿主轉譯蛋白質機制，藉此完成其生命週期並進行其致病機制。而在宿主方面，會因應產生一系列免疫反應來消除病毒的傳播。微核醣核酸(miRNAs)是近來發現的一類小片段、非蛋白編碼的核醣核酸，可藉由內源性RNA干擾方式來調控廣泛的生物功能，其中包括宿主與病毒的相互作用。於本論文中，我們觀察微核醣核酸於71型腸病毒感染前後表現量的差異，挑選了兩個在病毒感染過程中扮演重要角色的微核醣核酸 (miR-141與miR-146a)。miR-141為71型腸病毒感染細胞前後差異最顯著的微核醣核酸，可作用於轉譯起始因子4E，並關閉宿主的蛋白質合成，促進轉譯機制由cap-dependent轉譯轉換至cap-independent轉譯。miR-146a為另一個細胞受病毒感染而誘發的微核醣核酸，miR-146a可藉由3’非編碼區配對，進一步抑制兩個干擾素相關的蛋白質TRAF6和IRAK1表現。據研究指出，抑制TRAF6和IRAK1的表現會降低宿主干擾素反應。綜合以上結果，我們推測71型腸病毒可能是藉由調控上述兩個微核醣核酸來關閉宿主蛋白質合成並逃避宿主的抗病毒作用，進而有利於進行病毒的複製。 | zh_TW |
| dc.description.abstract | Virus processes complicated interactions with the host in virus infection. Virus relay on the host translation machinery to complete their life cycles and cause viral pathogenesis. In contrast, host utilizes immune system to eliminate virus spread. MicroRNAs (miRNAs) are a recently discovered class of small non-protein-coding RNAs that may act via endogenous RNA interference to govern a wide range of biological functions including host-virus interaction. In this thesis, we identified two miRNAs, miR-141 and miR-146a, which play significant roles in virus-host interactions. miR-141, the most up-regulated miRNA, targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis and promotes the switch from cap-dependent to cap-independent translation. miR-146a, the other up-regulated miRNA, suppressed two interferon-associated proteins, TRAF6 and IRAK1, through pairing to their 3’ untranslated regions. It was documented that suppression of TRAF6 and IRAK1 resulted in attenuation of host interferon responses. We hypothesized that EV71 might shutoff of host protein synthesis and escape host antiviral activity through regulation of the two miRNAs, and created a beneficial environment for its replication. | en |
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| dc.description.tableofcontents | 口試委員會審定書………………………………………………………i
Acknowledgements...……………………………………………………ii 中文摘要…………………………………………………………………iii Abstract…………………………………………………………………v Abbreviations…………………………………………………………vii Chapter 1 Introduction………………………………………………1 Chapter 2 Results………………………………………………………7 2.1 The expression of miR-141 is induced by enterovirus 71 infection………………7 2.2 eIF4E is a target of miR-141…………………………………9 2.3 MiR-141 involved in virus-induced translational switch…………………14 2.4 Silencing of miR-141 delays CPE occurrence and reduces virus production…………………15 2.5 miR-141 is partly regulated by EGR1………………………17 2.6 IRAK1 and TRAF6 are the targets of miR-146a……………22 2.7 AP1-associated signaling pathway was altered in EV71 infection…………25 2.8 miR-146a was upregulated by virus-infection-induced AP1………………………26 2.9 JNK inhibitor suppressed the EV71-infection-induced AP1 and further restored TRAF6 and IRAK1 expression……………28 Chapter 3 Discussions………………………………………………30 Chapter 4 Materials and Methods…………………………………38 4.1 Cell cultures and virus infection…………………………38 4.2 RNA extraction and miRNA profiling…………………………38 4.3 Oligonucleotide microarray analysis………………………39 4.4 Individual real-time RT-PCR…………………………………40 4.5 Western blot………………………………………………………41 4.6 Thiouridine incorporation assay……………………………41 4.7 Luciferase assay…………………………………………………42 4.8 Plasmid constructions…………………………………………42 4.9 Stable eIF4E-transfection of RD cells and antagomiR and siEGR1 transfections………………45 4.10 Metabolic labeling and immunoprecipitation……………45 4.11 Northern Blot……………………………………………………46 4.12 Plaque Assay……………………………………………………47 4.13 Chromatin immunoprecipitation assay………………………47 4.14 Interferon ELISA………………………………………………48 4.15 Sequence alignment analysis…………………………………49 4.16 JNK inhibitor treatment………………………………………49 4.17 Statistical analysis…………………………………………50 4.18 Signaling pathway analysis…………………………………50 Chapter 5 References….……………………………………………51 Chapter 6 Figures……………………………………………………65 Chapter 7 Tables……………………………………………………127 Chapter 8 Appendix…………………………………………………148 List of Figures Figure 1 Cap-dependent and cap-independent translation initiations………………65 Figure 2 Determination of Enterovirus 71 replication time……………66 Figure 3 Observation of the virus-induced CPE with different m.o.i.……………67 Figure 4 The proportion of virus-infected cells with different m.o.i.……………68 Figure 5 The miRNA expression profiles in virus-infected cell……………………69 Figure 6 miR-141 was induced by CVB3 and PV3 infections…70 Figure 7 Determination of miR-141 expression in EV71 infection……………71 Figure 8 Determination of Pri-miR-141 expression in EV71 infection……………72 Figure 9 Determination of newly synthesized miR-141 by thiouridine incorporation assay in EV71 infection…………73 Figure 10 miR-146a was induced by EV71 infection……………74 Figure 11 Predicted miR-141 binding site within the eIF4E 3’UTR………………75 Figure 12 The effect of miR-141 on endogenous eIF4E………76 Figure 13 Endogenous eIF4E targeted by miR-141………………77 Figure 14 The effect of EV71 infection on endogenous eIF4E…………………78 Figure 15 The effect of CVB3 or PV3 infection on endogenous eIF4E……………79 Figure 16 Determination of newly synthesized eIF4E in EV71 infection by pulse labeling assay………………………………80 Figure 17 Predicted sequence of the miR-141 binding site within the eIF4E 3’UTR……………81 Figure 18 The effect of miR-141 on the luciferase activity of reporter vectors with wild-type or mutant eIF4E 3’UTR……………82 Figure 19 The effect of miR-141 on the expression of eIF4E with wild-type or mutant 3’UTR………………………83 Figure 20 The effect of miR-141 on the expression of eIF4E with wild-type or mutant 3’UTR……………………………84 Figure 21 The effect of virus infections on the expression of V5-eIF4E fusion protein……………………………85 Figure 22 The reduction of eIF4E was restored by antagomiR-141……………86 Figure 23 Shutoff of host protein synthesis was delayed by antagomiR-141…………………87 Figure 24 miR-141 specifically attenuated Cap-dependent translation…………………88 Figure 25 Expression of viral proteins was suppressed by antagomiR-141…………………89 Figure 26 Expression of viral RNA was suppressed by antagomiR-141………………………90 Figure 27 Virus-induced CPE was attenuated by antagomiR-141..…………………………91 Figure 28 The effect of antagomiR-141 on virus infection-induced CPE..…………92 Figure 29 Virus production was attenuated by antagomiR-141……………………93 Figure 30 Reduction of virus production induced by antagomiR-141 was rescued by eIF4E siRNAs……………94 Figure 31 The effect of EV71 infection on endogenous eIF4E in neural cell……………95 Figure 32 The reduction of eIF4E was restored by antagomiR-141 in neural cells………………96 Figure 33 AntagomiR-141 reduces the virus production in neural cells……………97 Figure 34 Schematic organization of miR-141…………………98 Figure 35 Identification of the regulatory elements of miR-141..…………………99 Figure 36 Identification of regulatory elements of miR-141……………100 Figure 37 EGR1 was induced by EV71 infection………………101 Figure 38 EGR1 was induced by EV71 infection………………102 Figure 39 EGR1 binding sites were determined by chromatin immunoprecipitation assays……………………103 Figure 40 EGR1 was induced by CVB3 and PV3 infections……104 Figure 41 miR-141 and miR-200c located within the same pri-miR……105 Figure 42 miR-141 and miR-200c were under EGR1 regulation………………106 Figure 43 EGR1 activates miR-141 expression… ……………107 Figure 44 EGR1 siRNAs attenuate virus propagation…………108 Figure 45 The suppression of viral protein caused by EGR1 silencing is rescued by eIF4E silencing………………………109 Figure 46 Predicted miR-146 binding sites within the TRAF6 3’UTR…………110 Figure 47 Predicted miR-146 binding sites within the IRAK1 3’UTR…………111 Figure 48 The effects of EV71 infection on endogenous TRAF6 and IRAK1……112 Figure 49 The effects of miR-146a on endogenous TRAF6 and IRAK1…………113 Figure 50 The effects of miR-146a on the luciferase activity of reporter vectors with wild-type or mutant TRAF6 3’UTR……………………………………114 Figure 51 The effects of miR-146a on the luciferase activity of reporter vectors with wild-type or mutant IRAK1 3’UTR……………………………………115 Figure 52 The effects of miR-146a on the expression of TRAF6 and IRAK1 with wild-type or mutant 3’UTR……………116 Figure 53 c-jun and c-fos expressions were induced by EV71 infection………117 Figure 54 c-jun and c-fos expressions were induced by EV71 infection…………118 Figure 55 Schematic organization of miR-146a……………119 Figure 56 The effects of AP1 (c-jun/c-fos) on the luciferase activity of miR-146a promoter vectors…………120 Figure 57 The effects of AP1 (c-jun/c-fos) on endogenous miR-146a expression………………121 Figure 58 The effects of AP1 (c-jun/c-fos) on the luciferase activity of wild-type or mutant miR-146a promoter vectors……………………………………122 Figure 59 miR-146a promoter activity was regulated by EV71 infection…………123 Figure 60 The effects of JNK inhibitor on c-jun, c-fos, TRAF6, and IRAK1 expressions in EV71 infection……………124 Figure 61 Model for the regulatory role of miR-141 in enterovirus infection……125 Figure 62 The hypothetical model for the regulatory role of miR-146a in EV71 infection………………………………………126 List of Tables Table 1 Potential targets of miR-141…………………………127 Table 2 The expression levels of interferon α in antagomiR-141 transfectants………………130 Table 3 The altered expression of transcription factors in EV71 infection………………………131 Table 4 The expression levels of interferon α in siEGR1 transfectants………………132 Table 5 Potential hits of EV71 viral genome by EGR1 siRNAs…………………133 Table 6 Potential targets of miR-146a…………………………134 Table 7 The altered signaling pathways in EV71 infection………………144 Table 8 Primers list………………………147 | |
| dc.language.iso | en | |
| dc.subject | 干擾素反應 | zh_TW |
| dc.subject | 微核醣核酸 | zh_TW |
| dc.subject | 微核醣核酸141 | zh_TW |
| dc.subject | 微核醣核酸146a | zh_TW |
| dc.subject | 轉譯機制轉換 | zh_TW |
| dc.subject | MicroRNAs | en |
| dc.subject | miR-141 | en |
| dc.subject | miR-146a | en |
| dc.subject | translation switch | en |
| dc.subject | interferon responses | en |
| dc.title | 微核醣核酸於71型腸病毒感染中對病毒複製和宿主免疫反應的影響 | zh_TW |
| dc.title | The impacts of miRNAs on viral replication and host immune responses in enterovirus 71 infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 楊泮池(Pan-Chyr Yang),施信如(Shin-Ru Shih),高全良(Chuan-Liang Kao),張淑媛(Sui-Yuan Chang),俞松良(Sung-Liang Yu) | |
| dc.subject.keyword | 微核醣核酸,微核醣核酸141,微核醣核酸146a,轉譯機制轉換,干擾素反應, | zh_TW |
| dc.subject.keyword | MicroRNAs,miR-141,miR-146a,translation switch,interferon responses, | en |
| dc.relation.page | 150 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-01-18 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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| ntu-100-1.pdf 未授權公開取用 | 10.37 MB | Adobe PDF |
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