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

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)

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)