探討IFNγ mRNA 5端及3端非轉譯區的轉譯抑制訊息調控

Abstract

鼻之自然殺手細胞淋巴癌為與EB病毒相關且從鼻咽黏膜所引發之淋巴癌。雖然自然殺手細胞通常可以消滅被病毒感染的細胞，但目前仍尚未釐清為何病毒可以存活於自然殺手細胞中並會侵犯鄰近組織。EB病毒會轉錄出一些特定microRNA (miRNA) ，藉由這些miRNA，我們進而探討其在細胞中的調控機制。 在過去的實驗數中已知IFNγ轉譯抑制對於NK 細胞中的EB病毒影響重大。故我們利用微陣列 (micro array) 及qRT-PCR (quantitative RT-PCR) 的方式分析39個EB病毒miRNA 於EBV-positive 的YT及NK92細胞株。進而發現有12個EB病毒miRNA 於YT 細胞中的表現最為顯著, 然而在NK92細胞則未有明顯表現。並且，由於miRNA藉由與標的mRNA 3端非轉譯區 (3’ UTR) 部分或完全鹼基配對來調控轉譯進行，進而抑制標的基因的轉譯或者誘導標的mRNA的分解。為研究EB病毒的miRNA在自然殺手細胞是否會影響與IFNγ表現，首先，我們在IFNγ的3’ UTR進行9種基因依序剃除突變株，用西方墨點轉漬法 (western blot) 及螢光試驗 (luciferase assay) 在YT細胞中尋找miRNA在IFNγ的結合位。我們剔除的基因若為miRNA結合位，則western blot中 IFNγ蛋白表現或luciferase assay中的冷光表現會明顯增加。接著分析這些可能的結合位與YT細胞中表現較高的EB病毒是否相關，進而發現miR-BART20在IFNγ的3’ UTR上具結合位。並且，我們希望能建立起利用慢病毒 (lentivirus) 感染NK92細胞株的實驗系統以進一步研究miR-BART20在IFNγ的調控抑制。

Nasal NK-cell lymphoma (NNKCL) is an Epstein-Barr virus (EBV)-associated lymphoma arising from the nasal mucosa. Because NK cells usually destroy virus-infected cells, it’s unclear how the EBV can survive in NK cells and spread to nearby tissues. Our preliminary data showed that inhibition of IFNγ translation might be a critical factor in the survival of EBV inside NK cells. Furthermore, we performed quantitative RT-PCRs for 39 EBV-encoded miRNAs. Twelve EBV-encoded miRNAs had higher expressions in IFNγ-negative YT cells than in IFNγ-positive NK92 cells, suggesting the possibility of miRNAs in the regulation of IFNγ expression. Translation of mRNAs is usually regulated by elements in the 3’ or 5’ untranslated region (3’UTR/ & 5’UTR). Therefore, we constructed IFNγ mutants to identify sites critical for translational block directed by the miRNAs in YT cells. If the IFNγ mutant site is the target site for miRNAs, translation of the mutant IFNγ would not be blocked but would be increased. We constructed 9 stepwise deletions in the 3’UTR of IFNγ. With western blotting and luciferase assay, we found out that EBV encoded miR-BART20 might potentially target 3’ UTR of IFNγ. Currently, we are using a lentiviral transduction system to express EBV-encoded miR-BART-20 in NK92 cells, and to further investigate inhibition of IFNγ by this miRNA.