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研究生:賴琪婷
研究生(外文):Chi Ting Lai
論文名稱:利用蛋白質體學方法探討EBV-miR-BART18-5p之作用標的
論文名稱(外文):Study of ebv-miR-BART18-5p targets by proteomics approach
指導教授:余兆松陳華鍵
指導教授(外文):J. S. YuH. C. Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:94
中文關鍵詞:EB病毒
外文關鍵詞:EBV
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EBV(Epstein-Barr virus)的感染通常會導致許多疾病以及與許多癌症有密切的關聯,而其中包括好發於東南亞的鼻咽癌(nasopharyngeal carcinoma)。當EBV感染宿主細胞時,除產生其病毒蛋白外,亦會製造出microRNA (miRNA)。miRNA為有19~25個核苷酸所組成的RNA小分子,可結合在mRNA的3’UTR而抑制基因的表現。利用定量RT-PCR的方式偵測在鼻咽癌組織中ebv- miRNA的表現量,結果發現ebv-miR-BART18-5p (BART18-5p) 相較於其他ebv-miRNA表現量最高。為進一步研究BART18-5p在鼻咽癌中所扮演的功能,因此利用lentiviral expression system建立可穩定表現BART18-5p的HK1鼻咽癌細胞後,進一步利用iTRAQ-LC/MS/MS蛋白質體技術尋找差異表現的蛋白質。此實驗可定量到950個蛋白質,其中表現量上升者90個,表現量下降者75個 (mean ratio ≧1SD)。同時利用實驗室TargetScan分析BART18-5p可能的標的基因。結果顯示有9個表現量下降的蛋白質3’UTR有BART18-5p的結合位置。根據ContextScore、BART18-5p結合位置的數量與標的基因的3’UTR的長度,挑選5個可能為BART18-5p的標的基因:LUC7L2、DDX3X、TXNDC1、SUMO1、HMGA2,利用定量PCR以及luciferase assay驗證之,並以西方點墨法驗證SUMO1蛋白質於HK1/ BART18-5p細胞內表現情形時,亦可發現細胞內有特定蛋白質sumoylation的情形受到抑制。此外,同時利用microarray的分析結果找出1018個表現量差異的基因 (p-value < 0.05),以MetaCore分析BART18-5p所調控的基因網絡。發現BART18-5p可影響NF-κB 訊息傳遞,且NF-κB下游基因表現量也增加。其中BART18-5p的標的基因SUMO1可能參與負調控NF-κB訊息傳遞的角色。綜合以上,我們可利用蛋白質體學的方式尋找BART18-5p的標的基因,並推測BART18-5p可抑制SUMO1基因的表現,但SUMO1以及NF-κB訊息傳遞間的關係仍須進一步研究。
Latent Epstein-Barr virus (EBV) infection is closely associated with several cancers, including nasopharyngeal carcinoma (NPC). Some of the EBV-encode miRNAs have been found to influence the immune responses and apoptosis of the host, but the functions of most EBV miRNAs remain unknown. Using a stem-loop RT-PCR to quantify miRNA levels of EBV miRNAs in clinical tissues, we found that ebv-miR-BART18-5p (BART18-5p) was highly expressed in NPC samples. We used the lentiviral expression system to generate a cell line stably expressing BART18-5p and conducted proteomic and transcriptomic study to explore the biological function of BART18-5p. We employed a quantitative proteomic strategy (iTRAQ-LC-MS/MS) and quantified 950 proteins in control and BART18-5p cells. We found 90 up-regulated and 75 down-regulated proteins (mean ratio ≥ 1SD). We also use our in-house TargetScan algorithm and predicted potential targets for BART18-5p. Nine of the down-regulated genes were found to contain BART18-5p binding sites on their 3’UTR, suggesting that these genes could be direct targets of BART18-5p. SUMO1, one of the putative BART18-5p targets, was verified by real-time RT-PCR and luciferase assay. Western blot analyses revealed changes in SUMO1 targeting patterns in BART18-5p over-expressing cells. We used the Phalanx Human One Array to compare the expression levels of 19678 genes in control with BART18-5p over-expressing cells and identified 1018 differentially expressed (p-value < 0.05). These differentially expressed genes were analyzed by MetaCore to identify signaling pathways significantly regulated by BART18-5p. We found that the NF-κB signaling pathway and the expression level of several NF-κB downstream
genes were up-regulated in BART18-5p cells. SUMO1 is an up-stream inhibitor of NF-κB pathway. Our results suggest BART18-5p could regulate SUMO1, but the role of SUMO1 in BART18-5p mediated up-regulation of NF-κB need to investigation.
指導教授推薦書 ……………………………………………………… i
口試委員會審定書 ………………………………….…….………… ii
長庚大學博碩士紙本論文著作授權書 …………………………..… iii
誌謝 ……………………………………………….…………………… iv
中文摘要 …………………………………………………….………… v
英文摘要 …………………………………………………….………. vii
目錄 ………………………………………………..…………...……. ix
第一章 緒論…………………………………………………….…….. 1
1.1 Epstein-Barr Virus ……………………………………….…… 1
1.2 Epstein-Barr Virus genome ……...…….……………………….. 2
1.3 Epstein-Barr Virus-encoded microRNA ……………………… 3
1.4 MicroRNA 形成及作用 ……………………………………… 4
1.5 利用蛋白質體學分析miRNA的作用標的 ………………… 6
第二章 研究動機與目的…………………………………………….. 7
第三章 材料與方法…………………………………….…………. 8
3.1 細胞培養 ……………….………………………………… 8
3.2 載體製備 …………………………………………..……...... 8
3.3 Lentiviral expression system ……………………………….... 10
3.4 DNA轉染作用 (Transfection) ………………………………. 12
3.5 萃取RNA …………………………………………………… 12
3.6 反轉錄聚合酶連鎖反應 ……………………………...……. 13
3.7 即時定量聚合酶連鎖反應 …………………………...……. 13
3.8 穩定同位素標定之定量蛋白體技術...…….……………… 14
3.9 Reporter assay ………………………………….…………….. 15
3.10 西方點墨法 (western blot) ……………………………. 15
3.11 統計分析 …………………………………………………. 16
第四章 實驗結果 ……………………………….…………………... 17
4.1 利用lentiviral expression system將ebv-mir-BART18-5p 大量表現於HK-1、TW02及HEK293等細胞株 ………………...... 17
4.2以同位素標定蛋白體技術,分析在HK1/ebv-miR-BART18-5p細胞株內ebv-miR-BART18-5p所調控的差異性蛋白質…... 18
4.3 運用生物資訊分析ebv-mir-BART18-5p可能的標的基因… 20
4.4 以定量PCR檢測ebv-miR-BART18-5p標的基因之RNA表現 .... 21
4.5 利用Luciferase reporter assay 驗證ebv-miR-BART18-5p對於可能的標的基因之轉譯層級的影響 ……………….………. 21
4.6 利用西方點墨法偵測在HK1細胞中SUMO1蛋白質修飾化 (sumoylation) 的表現情形 …………………………….……. 22
4.7 利用microarray分析ebv-miR-BART18-5p標的基因SUMO1在HK1細胞中所參與的調控途徑 ……………………………. 24
第五章 討論 ……………………………………………………… 26
實驗結果圖表 …………………………...………………………… 34
參考文獻 ……………………………………………………….…..... 44
附錄 ………………………………………………………..…………. 46
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