Abstract
摘要:背景: 鼻NK 細胞淋巴瘤 (nasal NK-cell lymphoma, NNL) 是Epstein-Barr 病毒 (EBV)感染所引起的淋巴瘤。在NK 細胞 (cytotoxic NK cells) 中,轉錄因子T-bet 調控細胞激素IFNg (cytotoxic cytokine IFNg) 。EB 病毒編碼的miR-BART20-5p 不但會抑制T-bet 和IFNg 的轉譯 (Am J Path, 2013 & 2014),且會與miR-494-3p 及miR-142-3p共同調控PTEN-AKT-mTOR/Rictor 此一分子途徑 (Am J Path, 2015, underrevision)。此外,T-bet 的轉譯受T-bet mRNA 多個stem-loop 所調控,可能與核糖核蛋白複合體 (RNPs) 相關聯 (尚未發表)。目的:於小鼠模型中,以RNA pull-down 及siRNA Library,找出與T-bet 相關,可調控T-bet 轉譯之核醣核蛋白複合體 (RNPs)。實驗方法和預期結果1> 在T-bet+/EGFP 鼠中追蹤T-bet 的轉錄:我們已將內源性T-bet 編碼區替換成EGFP藉以追蹤EGFP+之NK 細胞。2> 追蹤T-bet 轉譯:表達ECFP-IRES-T-bet 的載體將被用於在EGFP+ 之NK 細胞中,並以ECFP 之表現監測T-bet 之轉譯。3> 以RNA pull-down 方法找尋T-bet 相關RNPs : 建構 (REV-peptide)-(REV-aptamer)- ECFP- IRES-T-bet 的載體,經RNA pull-down 後,透過質譜分析和RNA 定序作為找出T-bet 相關之RNPs。4> 以RNPs 作為T-bet 的活化劑或抑制劑:全基因體siRNA Library 將被用在ECFP−或者ECFP+ NK 細胞中,藉以辨別出T-bet 抑制劑或活化劑。5> NK 細胞發育:因為未成熟NK 細胞中T-bet 的轉譯會被T-bet 相關RNPs 阻斷。在小鼠或人類NK 細胞中的,T-bet 或和T-bet 相關的RNPs 表達模式,將被用來建立NK細胞發育的模型。6> NK 細胞淋巴瘤:藉由whole exome sequencing 的技術,比較有T-bet 轉譯抑制的NNL 和無T-bet 轉譯抑制的NNL 之間的差別。新的SNP, insertion, deletion 將被辨別出來,並探討與臨床和病理特徵上的相關性。意義:因為RNPs 包括RNA 結合蛋白質和非編碼RNA,我們希望找出在NK 細胞發育中可調控T-bet 轉譯之RNA 結合蛋白質或非編碼RNA,這將會修正當前的NK 細胞發育的模型及淋巴瘤分類,並可引進新的治療實體。
Abstract: Background: Nasal NK-cell lymphoma (NNL) is an Epstein-Barr virus (EBV)-infected lymphoma. Incytotoxic NK cells, the transcription factor T-bet controls IFNg, the primary cytotoxic cytokine. TheEBV-encoded miR-BART20-5p inhibits T-bet and IFNg (Am J Path, 2013 & 2014), and co-operate withmiR-494-3p and miR-142-3p in regulating a PTEN-AKT-mTOR/RICTOR pathway (Am J Path, 2015under revision). Significantly, T-bet translation is also regulated by multiple stem-loops of T-bet mRNAs,possibly in association with ribonucleoprotein particles (RNPs) (unpublished data).AIM: Characterization of T-bet-associated RNPs in a murine model of NK cell development and in NNLExperiments and expected results1> Tracking T-bet transcription in T-bet+/EGFP mice: The endogenous T-bet coding region has beenreplaced with EGFP to track EGFP+/CD122+ NK cells.2> Tracking T-bet translation: A vector expressing ECFP-IRES-T-bet will be used to monitor correlatedtranslation of ECFP and T-bet in the T-bet+/EGFP NK cells.3> RNA pull-down assay for T-bet-associated RNPs: A Rev-peptide-Rev-aptamer-ECFP-IRES-T-betplasmid will be constructed. RNA pull-down followed by mass spectrometry and RNA sequencing will beused to isolate T-bet-associated RNPs4> RNPs as T-bet activators or inhibitors: genome-wide siRNA screen will be used to identify T-betinhibitors or activators in ECFP- or ECFP+ NK cells.5> NK cell development: Expression profiles of T-bet and T-bet-associated RNPs in wild-type murine orhuman NK cells will be used to refine the current model of NK cell development, specifically, immatureNK cells with T-bet translation block.6> NK cell lymphoma: NNL with or without T-bet translation block will be compared. Novel SNPsinsertions, or deletions will be identified with whole exome sequencing to correlate with clinical andpathologic features.Significance:Because RNPs include both RNA binding proteins and non-coding RNAs, we expect to identify novelnon-coding RNAs critical in NK cell development via regulation of T-bet translation. This will revisecurrent classification of NNL and introduce new entities that require tailored therapeutic interventions.
Keyword(s)
T-bet
核糖核蛋白顆粒
非編碼RNA
鼻淋巴瘤
NK 細胞
T-bet
ribonucleoprotein particles
non-coding RNA
nasal lymphoma
NK cell