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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹世鵬 | |
dc.contributor.author | Chun-Ming Hsu | en |
dc.contributor.author | 徐君明 | zh_TW |
dc.date.accessioned | 2021-06-15T11:34:10Z | - |
dc.date.available | 2021-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49548 | - |
dc.description.abstract | 微小核醣核酸(miRNA)是一種新穎的微小非編碼核醣核酸,它在後轉錄的層次上藉由和目標基因的三端不轉譯區以鹼基配對的方式來調控各種下游基因的表現。某些微小核醣核酸異常的表現會扮演著腫瘤抑制因子的角色,例如let-7,若不正常表現將會導致人類發育異常及腫瘤生成。在我們的報導中NME4是一個高可性度的let-7的目標基因,在人類胚胎腎細胞、肝癌細胞中加入過量的let-7模擬物會使NME4蛋白質有減少的趨勢,相反地,在用let-7的反義股抑制住let-7後使NME4蛋白質有增加的趨勢。接著我們將NME4的三端不轉譯區選殖到帶有冷光酵素基因的載體中,並且測量在細胞中加入了let-7模擬物及抑制物後冷光活性的變化,我們發現了let-7模擬物會降低帶有NME4三端不轉譯區質體的螢光表現而用let-7反義股將let-7抑制住則會提高螢光表現,另外,在TargetScan軟體預測NME4三端不轉譯區上的let-7結合位進行突變卻能使此現象消失,因此綜合以上我們認為NME4會受到let-7的調控。 | zh_TW |
dc.description.abstract | MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs that regulate expression of various downstream genes at the post-transcriptional level, achieved through partial base-pairing between miRNAs and the target sequences at the 3’UTRs of mRNAs. Aberrant expression of certain miRNAs serving as tumor suppressors, such as let-7, leads to abnormal development and conduces to progression of human cancers. Here we report that the non-metastatic cells 4 (NME4) is a “high-confidence” let-7 target. The protein levels of NME4, examined by western blots, were down-regulated when excess amount of let-7 mimics were introduced into HEK293, Huh7 or HepG2 cells. By contrast, inhibition of let-7 by introducing antisense inhibitors increased the NME4 protein levels. We cloned the NME4 3’UTR into a luciferase vector and measured the luciferase activity of lysates prepared from the cells transfected with let-7 mimics or inhibitors. We found that let-7 mimics reduced the luciferase activity and let-7 inhibitors showed an opposite effect. Mutations of the putative let-7 target site at the NME4 3’UTR, predicted by the TargetScan miRNA target prediction software, eliminated the effects on the luciferase activity. Thus, we propose that NME4 is regulated by the let-7 miRNA. The biological implication of the let-7-NME4 regulation awaits further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:34:10Z (GMT). No. of bitstreams: 1 ntu-105-R03445124-1.pdf: 3286518 bytes, checksum: 899be97702eb3e3b0b7895da766470ab (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 #
誌謝 i 中文摘要 ii Abstract iii 目錄 iv Chapter 1 Introduction 1 1.1 microRNA的功能 1 1.2 let-7和目標基因在癌症中扮演的角色 1 1.3 預測let-7目標基因的模型 2 1.4 NME4可能為let-7的目標基因 3 1.5 NME4的功能 4 1.6 癌症中NME4和粒線體動態平衡的關係 5 Chapter 2 Materials and methods 6 2.1 西方墨點法 6 2.1.1 膠體製備 6 2.1.2 電泳、轉印及抗體雜和 6 2.2 北方墨點法 7 2.2.1 實驗流程 7 2.2.2 探針製備 8 2.2.3 探針引子序列 8 2.3 聚合酶連鎖反應 8 2.4 細胞培養 9 2.4.1 細胞株 9 2.4.2 細胞培養環境 10 2.5 細胞轉染 10 2.6 基因沉默 11 2.6.1 siRNA基因沉默 11 2.6.2 shRNA 基因沉默 11 2.7 Luciferase assay 12 2.8 細胞萃取液製備 13 2.9 let-7模擬物及抑制物 14 2.10 粒線體染色 15 Chapter 3 Results 16 3.1 細胞中NME4的表現可能和let-7的表現有關 16 3.2 let-7會影響NME4的表現 16 3.3 let-7會直接經由三端不轉譯區調控NME4 17 3.4 let-7調控NME4的路徑可能會影響肝癌細胞中粒線體的形態 18 Chapter 4 Discussion 20 4.1 let-7經由NME4 之3端不轉譯區域調控其基因表現 20 4.2 let-7對NME4 3端不轉譯區域之冷光表現有不同程度的調控 20 4.3 NME4 可能會經由let-7影響粒線體的形態 21 4.4 NME4調控粒線體的形態可能和癌症有關 21 4.5 NME4可能參與RAS網絡相關蛋白的調控 22 Chapter 5 Figures 23 Fig 1. NME4的表現可能和let-7的表現有關 23 Fig 2. let-7對NME4蛋白質表現的調控 25 Fig 3. let-7經由三端不轉譯區調控NME4 27 Fig 4. let-7對NME4的調控可能影響肝癌細胞粒線體的形態 30 Chapter 6 References 31 Chapter 7 Appendix 34 附圖一、以系統生物學之研究策略探究微小核醣核酸辨認其目標結合位之模型……. 34 附圖二、以帶有shRNA的lentivirus將目標基因敲落來測試NME4、SERF2、TRABD的抗體是否可以應用 35 附圖三、在人類細胞內過量表現let-7家族模擬物後觀察NME4、TRABD、SERF2的表現. 36 附圖四、psicheck2-1-TRIM71 3’ UTR map 37 附圖五、psicheck2-1-NME4 3’ UTR map 38 附圖六、psicheck2-1-NME4 3’ UTR(del) map 39 附圖七、psicheck2-1-NME4 3’ UTR(mut) map 40 附圖八、GFP-NME4 質體構築流程 41 附圖九、觀察帶有線狀或塊狀粒線體的Huh7細胞 44 附表 一、Dual Luciferase Assay原始統計數據 45 | |
dc.language.iso | zh-TW | |
dc.title | let-7微小核醣核酸調控核苷酸二磷酸激酶NME4基因表現 | zh_TW |
dc.title | Expression of nucleoside diphosphate kinase NME4 is regulated by the let-7 microRNA | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐立中,蔡欣祐 | |
dc.subject.keyword | 微小核醣核酸,核?酸二磷酸激?,粒線體, | zh_TW |
dc.subject.keyword | let-7,NME4,mitochondria, | en |
dc.relation.page | 51 | |
dc.identifier.doi | 10.6342/NTU201602961 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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