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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林中梧 | |
| dc.contributor.author | Wei-Ting Huang | en |
| dc.contributor.author | 黃偉庭 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:10:29Z | - |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-16 | |
| dc.identifier.citation | 1 Lin, T. C., Chen, S. U., Chen, Y. F., Chang, Y. C. & Lin, C. W. Intramucosal variant of nasal natural killer (NK)/T cell lymphoma has a better survival than does invasive variant: implication on loss of E26 transformation-specific sequence 1 (ETS-1) and T-box expressed in T cells (T-bet) with invasion. Histopathology 60, 287-295, doi:10.1111/j.1365-2559.2011.04086.x (2012).
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18537 | - |
| dc.description.abstract | Epstein-Barr病毒（EBV）感染常見於淋巴瘤疾病而鼻腔NK細胞淋巴瘤(NNL)是個與EBV病毒相關並由鼻黏膜中的細胞毒性NK細胞與T細胞衍伸而來。EB病毒編碼的miR-BART20-5P抑制T-bet和IFNg並間接抑制p53導致疾病發展的鼻腔NK細胞淋巴瘤（NNL）。
除此之外，我們尚研究EBV病毒如何藉由病毒本身的microRNAs來抑制IFN-γ-STAT1路徑以提升病毒的複製與主流的生長。在EBV−的Jurkat細胞當中，轉染miR-BART20-5p以及miR-BART8各別抑制了luciferase-IFN-γ-3’-UTR和luciferase-STAT1-3’-UTR的轉譯。在EBV+ IFN-γ表現弱/STAT1表現強的YT白血病細胞與IFN-γ表現強/STAT1表現弱的NK92細胞中，miR-BART20-5p與IFN-γ mRNAs或miR-BART8與STAT1 mRNAs在細胞內的相對表現量影響標的基因的表現。染色質免疫沉澱實驗顯示STAT1調節腫瘤抑制基因P53以及miR-let7a的轉錄。此外，使miR-BART8於YT細胞中過度表現或使miR-BART20-5p於NK92細胞中過度表現會抑制p53的表現而造成腫瘤對doxorubicin的抗藥性，這項實驗結果與前述實驗結果相符。於36位NNL的病人當中，miR-BART20-5p或miR-BART8的表現量與STAT1之表現呈現反比結果。除此之外，於46位NNL病人案例中，同時具有miR-BART20-5p和miR-BART8表現的一群NNL病人之p53 mRNAs均有下降情形且伴隨著疾病嚴重進展。因此，我們下了一個結論，EB病毒編碼的miR-BART20-5P和miR-BART8抑制干擾素IFN-γ-STAT1途徑與疾病進展的鼻NK細胞淋巴瘤息息相關 (已發表於2014年的AJP) (Appendix Figure 3)。 核醣核蛋白聚合物(RNPCs)可能調節著T-bet的轉譯過程。為了辨識這些核醣核蛋白聚合物，我們採用全基因體shRNA以分離並辨識那些可能誘導T-bet或是T-bet調節的標記的shRNAs。全基因體shRNAs同時也藉由病毒感染方式送入EBV+ 的YT-20-5p-ECFP-IRES-Tbet-EGFP細胞株，且在此實驗當中，我們以辨識出一些有表現ECFP(藍光)的clones。一些shRNAs已從分離出的clones當中定序出來且正進行進一步的確認實驗。 (關鍵詞：EBV病毒，鼻腔NK細胞淋巴瘤，NK細胞，T-bet，IFN-γ，miR-BART20-5p) | zh_TW |
| dc.description.abstract | Epstein-Barr virus (EBV) infection is frequently found in lymphomas, and Nasal NK/T-cell lymphoma (NNL) is an Epstein-Barr virus (EBV)-associated lymphoma derived from cytotoxic NK or T cells of the nasal mucosa. The EBV-encoded miR-BART20-5p inhibits T-bet and IFN-γ with secondary suppression of p53 and disease progression in nasal NK-cell lymphoma (NNL).
Furthermore, we investigated how EBV may have used miRNAs of viral origin to inhibit the IFN-γ-STAT1 pathway to facilitate viral replication and tumor growth. In EBV− Jurkat cells, transfection of miR-BART20-5p and miR-BART8 inhibited translation of luciferase-IFN-γ-3’-UTR and luciferase-STAT1-3’-UTR, respectively. In EBV+ IFN-γ weak/STAT1 strong YT leukemic cells and IFN-γ strong/STAT1 weak NK92 cells, relative endogenous levels between miR-BART20-5p and IFN-γ mRNAs or between miR-BART8 and STAT1 mRNAs determined expression of the targets. Chromatin immune precipitation studies showed that STAT1 regulates the transcription of the tumor suppressor TP53 (encoding p53) and miR-let7a. Consistent with these findings, overexpression of miR-BART8 in YT cells or of miR-BART20-5p in NK92 cells inhibited p53 and increased resistance to doxorubicin. In 36 NNLs, the levels of miR-BART20-5p or miR-BART8 correlated inversely with the expression of STAT1. Additionally, in 46 NNLs, expression of both miR-BART20-5p and miR-BART8 identified a group of NNLs with decreased p53 mRNAs and evidence of disease progression. We conclude that miR-BART20-5p and miR-BART8 cause progression of nasal NK-cell lymphomas through inhibition of the IFN-g-STAT1 pathway (Am J Path, 2014) (Appendix Figure 3.). Multiple ribonucleoprotein complexes (RNPCs) might regulate T-bet translation. To identify these RNPCs, we have used genome-wide shRNA library to isolate and identify the shRNAs which can induce T-bet or T-bet-regulated markers. The library was also transduced into EBV+ YT-20-5p-ECFP-IRES-Tbet-EGFP cells, and ECFP positive clones were identified. Several shRNAs have been sequenced in the isolated clones and processed for further confirmation. (Key words：EBV, Nasal NK/T-cell lymphoma (NNL), NK cell, T-bet, IFN-γ, miR-BART20-5p) | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:10:29Z (GMT). No. of bitstreams: 1 ntu-103-R01444002-1.pdf: 6703312 bytes, checksum: bf7ddee9beab7aeb155ef4c331fe553c (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 致謝 ……………………………………………………………………...................i
目錄 …………………………………………………………………….....................ii 中文摘要 ……………………………………………………………….....................1 Abstract …………………………………………………………………....................2 Chapter 1: Introduction …………………………………………………....................4 1.1 Epstein-Barr virus-encoded miR-BART20 and miR-BART8 in Nasal NK cell lymphoma………………………………………………………………….....4 1.2 Genome-Wide DECIPHER shRNA Library ………………………………….7 1.3 The model of miRNA translational repression………...…...………………….7 1.4 Potential T-bet-regulated-selection markers…….……………………………..8 1.5 Library screening to identify multiple ribonucleoprotein complexes (RNPCs)... ………………………………………………………………………………..11 Chapter 2: Materials and Methods …………………………………………………13 2.1 Chemicals ………………..………………………………………………….13 2.2 Machines and Kits ………………………………………………………13 2.3 Plasmid constructs ………………………………………………………14 2.4 Cell lines and stable cell lines …………………………………………15 2.5 Western Blotting ..……………………………………………………17 2.6 Amplification of the library and viral production ………………………….17 2.7 Transduction of the library into YT cells .......……………………………18 2.8 Flow cytometry ……………………………………….…….……………18 2.9 Cell Sorting …………………………….....…………………………………21 2.10 PCR ………………………………………………………………………...21 2.11 TA cloning of the library ...............................................................................22 2.12 Colony PCR ………………………………………………………………22 2.13 Real-time RT-PCR …………………………………………………………23 2.14 Sequences from colony PCR ………………………………………………24 2.15 Data analysis ……………………………………………………………24 Chapter 3: Results …………………………………………………………………..25 3.1 EBV-Encoded miR-BART20-5p and miR-BART8 Inhibit the IFN-γ-STAT1 Pathway Associated with Disease Progression in Nasal NK-Cell Lymphoma... ……………………………………………………………………………….25 3.2 Construction of the library in YT cells …………………...…………………25 3.3 Intra-cellular staining for T-bet in a YT-BART20-library: miR-BART20-5p suppresses endogenous T-bet in YT cells cells transduced with a shRNA-Decipher library and tarnfected with miR-BART20-5p…………....26 3.4 Double intra-cellular staining for T-bet and IFN-γ from YT cells transduced with the shRNA Library: isolation of rare cells ..............................................26 3.5 Identify potential T-bet-regulated markers in YT cells: T-bet does not induce CXCR3, CD44, CD94, or IL12RB2 in YT cells ……………………............27 3.6 Selection for T-bet-independent marker, CD44: Failure to enrich after 4 rounds ………………………………………….……………………………27 3.7 Array and qRT-PCR for potentialT-bet-dependent markers: WDFY4 is a weakly T-bet-regulated surface marker ……………………………………28 3.8 IL12 plus IL18 does not up-regulate an IFNg promoter-dsRed construct in YT cells ……………..……………………………………………….…………..28 3.9 IFNg promoter-dsRed-EGFP in YT and NK92 cells: Inconclusive for a T-bet driven IFNg promoter ………………………………......………………….29 3.10 Transfection of plasmid IFNg-promoter-dsRed-EGFP or plasmid Tbet-IFNg- promoter-dsRed-EGFP into YT cells: over-expression of T-bet might up-regulate the IFNg-promoter ………………..…………………………...29 3.11 20-5p-dsRed-IRES-Tbet-EGFP and 20-5p-dsRed-IRES-Tbet-3’UTR -deletion-EGFP in YT cells …………………………………………….….30 3.12 Plasmids 20-5p-ECFP-IRES-Tbet-EGFP and 20-5p-ECFP-IRES-Tbet- 3’UTR-deletion- EGFP may be used to screen the shRNA library in YT cells …………….................................................................……………….31 3.13 Conclusions ………………..………………………………………………31 Chapter 4: Discussion ……………….………………………………………………33 Figures and Tables …………………………………………………………………..38 Reference ……………………………………………………………………………56 Appendix ……………………………………………………………………………68 | |
| dc.language.iso | en | |
| dc.title | 以全基因體shRNA篩選能於YT細胞誘發受T-bet調控的標記之微RNA | zh_TW |
| dc.title | Genome-wide shRNA screen in YT cells for induction of T-bet- regulated markers | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡錦華,周綠蘋,陳惠文,黃聖懿 | |
| dc.subject.keyword | EBV病毒,鼻腔NK細胞淋巴瘤,NK細胞,T-bet,IFN-γ,miR-BART20-5p, | zh_TW |
| dc.subject.keyword | EBV,Nasal NK/T-cell lymphoma (NNL),NK cell,T-bet,IFN-γ,miR-BART20-5p, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2014-08-18 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 病理學研究所 | zh_TW |
| 顯示於系所單位： | 病理學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-103-1.pdf 未授權公開取用 | 6.55 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。