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研究生:吳宗凱
研究生(外文):吳宗凱(Chung kai Wu)
論文名稱:MicroRNAmiR-127在鼻咽癌中之功能性探討與其標的基因的找尋
論文名稱(外文):Functional characterization and target exploration of microRNA miR-127 in Nasopharyngeal Carcinoma
指導教授:鄧致剛鄧致剛引用關係
指導教授(外文):Petrus Tang
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:63
中文關鍵詞:miR-127NPCTargetfunctional
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microRNA(miRNA)是一種長度約19~25個核苷酸的內生性小RNA分子,藉由結合在mRNA的3’UTR而抑制基因的表現。過去文獻證明miRNA與細胞的生長以及癌症的形成扮演重要的角色。
本實驗室建立定量Q-PCR的分析平台,運用此分析平台分析270個miRNA在NPC檢體中的表現情形,發現Has-miR-127(miR-127)在鼻咽癌檢體相對於正常的檢體其表現量較低。除此之外,相對於較正常的NPC細胞,在NPC相關細胞株中也發現到miR-127其表現量也是下降。利用UCSC Genome Browser website 分析miR-127的promoter,結果發現其上游大約 500bp的位置有CpG island ,顯示miR-127的調控可能受DNA甲基化所影響。因此我們在HK1細胞中加入5-aza-2’- deoxycytidine (5-Aza-CdR)之後,也觀察到miR-127的表現量增加。這說明在NPC中miR-127可能受到DNA甲基化的調控。
為了研究miR-127在癌症形成過程中所扮演的角色以及可能調控的基因,我們將miR-127送入HK1使其大量表現,再利用MTS assay偵測細胞生長的速度,用microarray及生物資訊上的分析尋找miR-127可能調控的基因,最後用Q-PCR以及Reporter assay驗證所找到的標的基因。實驗結果發現miR-127大量表現會使HK1細胞生長趨於緩慢,顯示miR-127會調控細胞的生長。綜合microarray及生物資訊分析的結果,發現了4個可能的標的基因ARF1、DHRS2、PRKCZ、TLK2。將這些標的基因經由Q-PCR和Reporter assay驗證之後,發現PRKCZ、TLK2的表現確實會受到miR-127的抑制。miR-127是否可能藉由調控這幾個標的基因而影響細胞生長的速度,仍需用其他實驗來證明。
MicroRNAs (miRNA) are small endogenous noncoding RNAs of approximately 22 nucleotides (nt) in length. MicroRNAs usually bind to partially complementary sites in the 3’-untranslated region (3’UTR) of mRNA targets and either repress translation, induce degradation or deadenylation of their targets. Aberrant expression of miRNAs has been observed in many human disorders, including cancer.
To study the roles of miRNA in the pathogenesis of nasopharyngeal carcinoma (NPC), we characterized the expression profile of 270 miRNAs in nasopharyngeal carcinoma (NPC) clinical tissues and identified a set of miRNAs differentially expressed in NPC. MicroRNA hsa-miR-127 is down-regulated in NPC tissues. Compared to normal nasopharyngeal epithelial cell, expression level of has-miR-127 is also reduced in NPC cell lines. We identified CpG islands in the promoter region of hsa-miR-127 using the UCSC Genome Browser website. Treatment with 5-aza-2’-deoxycytidine(5-Aza-CdR) restored the expression of hsa-miR-127, suggesting that promoter methylation may regulate the expression of hsa-miR-127. It may provide a regulatory mechanism for has-miR-127 expression. In order to understand the role of hsa-miR-127 in NPC, we overexpressed hsa-miR-127 in HK1 cells and observed a reduction in the growth rate of HK1 cells. These results suggest that hsa-miR-127 may function as a tumor suppressor gene by inhibiting the growth of tumor cell.
In order to explore the target genes of hsa-miR-127 in NPC, we conducted microarray assay to analyze mRNA expression level in miR-127-overexpressing HK1 cells. We also use TargetScan to predict potential targets of hsa-miR-127. We found 4 genes, ARF1, TLK2 , PRKCZ, DHRS2, as candidate targets for hsa-miR-127. We validated the expression level of these target genes in cells overexpressing hsa-miR-127 by Q-PCR. Reporter gene assay confirmed that the 3’UTR of these target genes indeed contain regulation sites for hsa-miR-127. These target genes may be regulated by hsa-miR-127 and may play some role in the growth of tumor cell.
英文摘要………………………………………………………………2

中文摘要………………………………………………………………4

壹、背景介紹…………………………………………………………6

貳、研究動機與目的…………………………………………….…..14

叄、材料方法……………………………………………………...…15

肆、實驗結果…………………………………………………………25

陸、實驗結果讨論……………………………………………………32

柒、圖表………………………………………………………………36

捌、參考資料…………………………………………………………51

玖、附錄………………………………………………………………56
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