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標題: | 探討 YT 細胞株中調控 T-bet 之 microRNA Searching for microRNAs that regulate T-bet in YT cell line |
作者: | Ying-Chi Chen 陳英奇 |
指導教授: | 林中梧(Chung-Wu Lin) |
關鍵字: | microRNA,T-bet,後轉錄層面調控,, microRNA,T-bet,post-transcriptional control, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 在T輔助細胞(Th)中,特異性的細胞激素與轉錄因子經由特定的訊號傳遞路徑,來驅動T輔助細胞亞群的發育。第一型(Th1)和第二型(Th2)T輔助細胞之分化已經被廣泛的研究。轉錄因子T-box expressed in T cells (T-bet)與GATA binding protein 3 (GATA-3)分別在Th1及Th2細胞發育上扮演決定性的角色。這些具有Th細胞亞群特異性的轉錄因子,藉由活化特定細胞激素基因的啟動子,來調控趨化Th1或Th2細胞的細胞激素之表現。由於自然殺手細胞的分化尚未被非常清楚的定義,因此我們將T輔助細胞之分化觀念延伸至自然殺手細胞,進而以自然殺手細胞株 (NK92和YT) 為模式,探討T-bet及GATA3在自然殺手細胞發育過程中的調控。
我們由微陣列分析 (microarray) 及西方墨點轉漬法 (Western blotting) 的結果發現,在YT細胞株中,儘管T-bet 在mRNA的表現量很高,但在protein表現方面則明顯的降低,此結果暗示T-bet在YT細胞株中具有轉譯層面的調控;另外,藉由質譜儀 (mass spectrometry) 及西方墨點轉漬法發現T-bet可能具有isoforms。由這些初步研究的成果,讓我們著重於T-bet基因後轉錄調控機制研究。mRNA的轉譯通常是藉由它們5’或3’非轉譯區域 (UTR) 來進行調控。首先,我們構築不同的T-bet突變株,證明在YT細胞株中,T-bet mRNA藉由5’ UTR上推定的pseudoknot結構來進行轉譯抑制;另ㄧ方面,我們發現刪除3’UTR (Δ3’UTR-YT)的突變株,T-bet的表現有顯著的上升,這個結果暗示T-bet的3’UTR在轉譯調控上扮演著重要的角色。 MicroRNA (miRNA)藉由與標的mRNA 3’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。為了檢視T-bet的表現是否受到miRNA所調控,我們先利用miRNA資料庫預測可能標的在T-bet mRNA 3’非轉譯區的29個Homo sapiens miRNA,並以定量反轉錄聚合酶鏈式反應 (qRT-PCR) 來分析它們在YT,Δ3’UTR-YT,和NK92細胞株中的表現。我們發現了11個Homo sapiens miRNA在YT與NK92細胞株之間有表現的差異,其中miR-593*在YT細胞株中被顯著誘導表現,而miR-133a在YT細胞株的表現則是被顯著的抑制。由於這些細胞株皆為EBV-positive的細胞,所以我們同時也分析39個EBV miRNA的表現,並發現在表現上有差異的12個EBV miRNA,它們在YT細胞株的表現都遠大於NK92細胞株。最近已有研究顯示,病毒產生的miRNA可能具有免疫調控的功能,因此我們推測這些有差異的miRNA在T-bet基因後轉錄調控上很可能具有重要的功能,值得在未來更進一步地深入探討研究。藉由研究結構或miRNA作用在T-bet表現上的調控機制,將會幫助我們了解基因調控的複雜模式以及NK細胞發育的調控。 Cytokines and transcription factors specific to the T helper (Th) cell lineages are crucial for driving the development of Th cell subsets via specific signaling pathways. The concept of type I vs. type II differentiation was extensively studied in Th cells. Two transcription factors, T-box expressed in T cells (T-bet) and GATA binding protein 3 (GATA-3), function as master regulators for the development of Th1 and Th2 cells, respectively. These subtype- specific transcription factors of Th cells involved in Th1- and Th2- specific cytokines expression by activating cytokine gene promoters. Because the differentiation of NK cells has not been well defined, we extended the concept of T cell differentiation to NK cells. For this reason, we used YT and NK92 lymphoma cell lines of NK cell origin to examine the role of T-bet and GATA3 in development process of NK cells. To investigate the role of transcription factors, T-bet and GATA3, in differentiation process of NK cells, we analyzed gene expression profiles of NK-92 and YT cell lines by microarray and Western blotting. We found that T-bet was obviously weak in the YT cell line despite strong mRNA expression. The expression pattern suggested that T-bet might be controlled at the level of translation in the YT cell line. In addition, we also performed Mass Spectrometry/MALDI-TOF and antibody mapping, and the results showed that T-bet could have isoforms. The preliminary results hinted that we should focus our research on posttranscriptional control of T-bet. The translational control of mRNA is usually regulated by elements in their 3’ or 5’-untranslated region (3’UTR/5’UTR). At first, we utilized different constructs of T-bet mutants to verify the translational block directed by the putative RNA pseudoknot of 5’UTR in YT cells; on the other hand, we found that T-bet protein was obviously up-regulated in the construct of 3’UTR deletion stable clone (Δ3’UTR-YT). The result suggested that the 3’UTR of T-bet plays an important role in the translational control. MicroRNAs (miRNAs) regulate gene expression by partially or completely base-pairing with the 3’UTR of their target mRNAs and repressing gene translation or inducing mRNA degradation. To examine the possibility of T-bet regulation by miRNAs, we searched the miRNA database for potential miRNA candidates targeting the 3’UTR of T-bet, and 29 Homo sapiens miRNAs were predicted as putative regulators of T-bet. We performed qRT-PCR to identify significant miRNAs whose expression was the most significant in YT, Δ3’UTR-YT and NK92 cell lines. We found 11 Homo sapiens miRNAs with significant expression diversity between YT and NK92 cells. miR-593* was up-regulated most and miR-133a was down-regulated most in YT cells. Moreover, we also analyzed the expression patterns of 39 EBV miRNAs because all the cell lines are EBV-positive. Interestingly, we found that each of the 12 significant EBV-miRNAs was much higher in YT cells than in NK92 cells. Recent studies have shown that viral-encoded miRNA may invole in the immunomodulatory mechanism. The further work will be done to elucidate the roles of these significant miRNAs in the process of posttranscriptional regulation of T-bet. The study of the structure-mediated and miRNA-directed T-bet regulation will be helpful for us to understand the highly complex pattern of gene regulation the development of NK cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42367 |
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