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研究生:陳建宇
研究生(外文):Chien-Yu chen
論文名稱:以YT cells作為鼻NK細胞淋巴癌模型探討BCL11B 抑制T-bet mRNA 轉譯
論文名稱(外文):BCL11B inhibits translation of T-bet mRNA in YT cells: a model for nasal NK-cell lymphoma
指導教授:林中梧
指導教授(外文):Chung-Wu Lin
口試日期:2017-07-18
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:病理學研究所
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:2017
畢業學年度:105
語文別:英文
論文頁數:59
中文關鍵詞:鼻自然殺手細胞淋巴癌EBV病毒T-bet 轉錄因子B細胞淋巴癌11BGAIT-like結構
外文關鍵詞:Nasal NK-cell lymphomaEpstein-Barr virusT-betBCL11BGAIT-like domain
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鼻NK細胞淋巴癌(NNL) 是與EB病毒(EBV)有相關的NK细胞淋巴癌,而T-bet是個重要的轉錄因子,會影響毒殺T細胞或NK細胞interferon-gamma (IFN-γ) 的產生,EB 病毒編碼的miR-BART20-5P 抑制T-bet 而誘導鼻NK 細胞淋巴癌對組織的侵入性。YT cells 是人類EBV+ NK-like淋巴癌的細胞株,為了研究miRNA抑制T-bet轉譯的機制,我們用genome-wide shRNA library來篩選YT cells的方式找到BCL11B shRNA會誘導T-bet表現,之後將Flag-BCL11B-EGFP的表現質體用轉染的方式讓YT cells大量表現BCL11B,透過流式細胞分選儀分選出有EGFP的YT cells進行Western blotting分析,發現其T-bet蛋白表現量比EGFP negative的YT cells低,然而T-bet mRNAs並無顯著差異,接著在luciferase assays中我們發現BCL11B會對T-bet抑制是透過結合在T-bet 3’-UTR,而藉由序列比對分析更進一步發現抑制的結合區可能是位在T-bet 3’-UTR 的GAIT-like domains,之後經由RT-PCR分析來證實GAIT-like element-G4會與BCL11B的zinc finger domains作用。除此之外,我們透過YT cells 來表現His-EGFP-BCL11B、 RRE-T-bet-mRNA, 以及Flag-Rev-EGFP,利用Rev-RRE的作用再次證實BCL11B確實與T-bet mRNA的作用,最後我們合成GAIT-like elements 以及Zinc finger peptides透過pull down assay來計算出dissociation constant (Kd),這些結果將來會用在設計miRNA 或 peptide類似物或者是zinc finger抑制劑透過干擾BCL11B來減緩NNL的侵入性。
Nasal NK-cell lymphoma (NNL) is an Epstein-Barr virus (EBV)-associated aggressive lymphoma of NK cell origin, endemic in Taiwan. T-bet is the key transcription factor involved in interferon-gamma (IFN-γ) production by cytotoxic T or NK cells. EBV-encoded miR-BART20-5p inhibits T-bet translation and induces an aggressive and invasive behavior of NNL. The YT cell line is a human EBV+ NK-like lymphoma cell line. To investigate the mechanisms of miRNA-mediated T-bet translation inhibition, we transduced a genome-wide shRNA library into YT cells and found that the BCL11B shRNA could up-regulate T-bet expression. An expression vector for Flag-BCL11B-EGFP was transfected into YT cells. After sorting, EGFP positive YT cells had lower expression of T-bet than EGFP negative YT cells by Western blotting, in spite of similar levels of T-bet mRNAs by real-time RT-PCR. Using luciferase assays, we confirmed BCL11B interacts with 3’-untranslated region (UTR) of T-bet. Further analysis showed the presence of GAIT-like domains in the T-bet 3’-UTR. Real-time RT-PCR of precipitated lysate from YT cells overexpressing the zinc fingers of BCL11B confirmed interactions between interferon-gamma-activated inhibitor of translation (GAIT) -like elements and zinc finger domains of BCL11B. Finally, we used synthetic GAIT-like elements and zinc finger peptides in a pull-down assay to obtain a dissociation constant. The data will be used to design miRNA or peptide mimics and zinc finger inhibitor that may ameliorate the invasive behavior of NNL through interfering with BCL11B.
Contents I
致謝 III
摘要 IV
Abstract V
Chapter 1: Introduction 1
Chapter 2: Materials and Methods 5
2.1 Plasmid constructs and synthesis of RNA and peptides 5
2.2 Synthesis of G4 and peptide 5
2.3 Cell lines and stable cell lines 5
2.4 Cell sorting and flow cytometry 7
2.5 Western Blotting 7
2.6 Luciferase assay 9
2.7 Real-time RT-PCR 9
2.8 Ni-NTA magnetic beads 10
2.9 G4 RNA pull-down assay by BCL11B or zinc fingers after crosslink (Fig 4C) 11
2.10 G4 RNA pull-down assay by synthetic peptide without crosslink (Fig 5) 12
Chapter 3: Results 14
3.1 Five siRNAs from the genome-wide shRNA Library induce T-bet expression. 14
3.2 BCL11B and TRIM71 inhibit T-bet expression. 14
3.3 Localization of BCL11B and TRIM71 in YT cells. 15
3.4 BCL11B inhibit the luciferase via T-bet 3’-UTR. 15
3.5 GAIT-like elements and BCL11B zinc fingers inhibit T-bet translation. 16
3.6 G4 RNA interacts with the sixth zinc finger of BCL11B 16
3.7 His-EGFP-BCL11B co-precipitates Flag-Rev-EGFP via RRE-T-bet mRNA. 17
Chapter 4: Discussion 19
Chapter 5: Conclusion 24
Figures 25
Reference 39
Appendix 48
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