2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658616摘要：鼻自然殺手細胞淋巴癌與EB病毒相關，研究顯示抗原受器之局部分布(KIR repertoire)和CD94只表現影響是否有較佳之預後，但其機制和EB病毒感染的重要性仍不清楚。EB病毒表現EBER和大量的miRNA，其中EBER在霍金氏淋巴癌中可經抑制p21cip1/waf1，而產生抗藥性，但大部分的miRNA功能尚不清楚。鼻自然殺手細胞淋巴癌目前已知有非侵入性和侵入性兩種亞型。自然殺手細胞之成熟需要轉錄因子T-bet、CD94和interferon gamma (IFNg)。由於EB病毒miR-BART-20抑制T-bet和CD94，使得鼻自然殺手細胞淋巴癌無法成熟，且較更具有侵略性。此外，因T-bet調控p53和IFNg，T-be被抑制後，將去除tumor-suppressing p53和antiviral IFNg，故引發鼻自然殺手細胞淋巴癌的侵略行為。 我們將以miR-BATRT20-5p抑制T-bet這一結論為範例，分析EB病毒所有miRNAs的結合位（mRNA target），及其病理意義。 第一部分：全基因體篩選 genome-wide screens 1> 以cDNA library找尋miR-BART20的結合位：透過受miRNA調控藥物感受性之cDNA library，篩選出miR-BART20-5的所有結合位。 2> 以siRNA library找尋調節T-bet表現的siRNA：轉殖 siRNA library至以miR-BART20抑制T-bet的YT細胞中，藉此找尋可誘導出T-bet表現的siRNA。 3> 以miRNA library篩選出抑制T-bet表現的miRNA: 轉殖 miRNA library至表現T-bet的NK92細胞中，藉此找尋可抑制T-bet表現的miRNA。 第二部分：生化上及生物上的確認 1> miRNA-induced alterations in mRNA profiles：miRNA將轉殖進未成熟之YT細胞或成熟NK92細胞中，隨後進行mRNA profiling或proteomic screens。 2> 生物資訊和生化方法：預測結合位及剔除mRNAs的3’UTRs來做確認，並將 miRNAs和mRNAs以補償性變異證明結合位（compensatory mutagenesis for target confirmation）。 3> 功能探討：透過 antisnse miRNAs 和突變的結合位 (mutant target sites) 來作探討。 4> 動物模式：a) 將以轉殖miR-BART20的YT或anti-miR-BART20的NK細胞送入nude mice藉以找尋miRNA的重要性和作用標的。b) 從transgeneic mice中將目標基因剔除藉以探討鼻自然殺手細胞淋巴癌的致病機轉。 因我們已有10年下來的 pilot series，本計劃將不會使用台灣的臨床檢體，我們將和 MD Anderson Cancer Center 合作建立verification series。本計劃著重分子間的交互作用，可望進而找尋出可有效治療鼻自然殺手細胞淋巴癌的療法。並應用於其他EB病毒相關的疾病，例如霍金氏淋巴癌(Hodgkin lymphoma)、勃氏淋巴瘤(Burkitt)及移植後淋巴增生疾病(PTLDs)。<br> Abstract: Nasal NK cell lymphoma (NNKCL) is associated with Epstein-Barr virus (EBV). We showed that NNKCL has a restricted KIR repertoire (Am J Path, 2001) and CD94 is associated with a better prognosis (Blood, 2003). The mechanism of the association and the significance of EBV infection are unclear. EBV encodes a small RNA, EBER, and abundant miRNAs. EBER suppresses p21cip1/waf1 in drug-resistant Hodgkin lymphoma (Molecular Cancer, 2010, 9,32), but functions of most miRNAs are unknown. We reported non-invasive and invasive NNKCLs (Histopath, 2012). Maturation of NK cells requires transcription factor T-bet, CD94, and interferon gamma (IFNg). Because EBV-encoded miR-BART-20 inhibits T-bet, NNKCLs that express miR-BART-20 are immature and CD94-, tend to be invasive and more aggressive (Am J Path, 2012, revised). In addition, miR-BART20 suppresses p53 and IFNg, secondary to T-bet inhibition. Removals of both tumor-suppressing p53 and antiviral IFNg cause the aggressive behavior of NNKCL (See Preliminary data). We will clarify targets and functions of all EBV-encoded miRNAs, using inhibition of T-bet by miR-BATRT20-5p to illustrate our approaches. PART I: genome-wide screens 1> Direct cDNA library screens for miRNA targets: A cDNA library, with expression of cDNA linked to miRNA-controlled drug-sensitivity, will be used to screen all targets of miR-BART20-5p. 2> Reverse siRNA library screens for protein regulators of target translation: A siRNA library will be transfected into YT cells, which have T-bet inhibited by miR-BART20. We will screen for siRNAs that may induce T-bet. 3> MiRNA library screens for miRNA-interactions: A miRNA library will be transfected into NK92 cells, which express T-bet, and miRNAs that inhibit T-bet will be identified. PART II: Biochemical and biological confirmation 1> MiRNA-induced alterations in mRNA profiles: MiRNAs will be transfected into immature YT or mature NK92 cells of NK origin, followed by mRNA profiling or proteomic screens 2> Bioinformatic and biochemical approaches: Prediction of binding sites and confirmation through deletions of 3’UTRs of mRNAs, and compensatory site-directed mutagenesis of miRNAs and mRNAs 3> Functional studies: through antisnse miRNAs and mutated targets in cell lines 4> Animal model: a) Transplantation of transfected YT or NK cells into nude mice to identify significances of miRNAs or targets, b) Deletion of the targets in transgeneic mice to study pathogenesis of NNKCL. As we have a pilot series through 10-years’ studies, clinical specimens from Taiwan will not be used. Rather, a verification series will be sought through Prof Medeiros, the PI’s mentor (Extranodal NK/T-cell Lymphoma: A Report of 73 Cases at MD Anderson Cancer Center. Am J Surg Path, 2013). We focus on detailed molecular interactions in order to yield a therapeutic agent for NNKCL and possibly other EBV-related diseases, such as Hodgkin lymphoma, Burkitt, and PTLDs.