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Title: | 以YT cells作為鼻NK細胞淋巴癌模型探討BCL11B 抑制T-bet mRNA 轉譯 BCL11B inhibits translation of T-bet mRNA in YT cells: a model for nasal NK-cell lymphoma |
Authors: | Chien-Yu chen 陳建宇 |
Advisor: | 林中梧(Chung-Wu Lin) |
Keyword: | 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11B,GAIT-like結構, Nasal NK-cell lymphoma,Epstein-Barr virus,T-bet,BCL11B,GAIT-like domain, |
Publication Year : | 2017 |
Degree: | 碩士 |
Abstract: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67156 |
DOI: | 10.6342/NTU201702705 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 病理學科所 |
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ntu-106-1.pdf Restricted Access | 1.86 MB | Adobe PDF |
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