臺灣博碩士論文加值系統

MicroRNAs ( miRNAs )是長度約22 nt的微小調控RNAs，其源自於似髮夾型態的前驅miRNAs，並且藉由結合到標靶基因的mRNA來阻止其轉譯作用，扮演著基因調控的角色。
本研究中，首先試著去找出位於啟動子區域的miRNA基因，並搜尋與基因子位置與CpG islands有關的miRNA基因。發現miRNA基因中有高比率位於CpG islands的1000 bp之內（約16.0 %）。並發現有10個miRNA基因是位於起動子區域內。其次，本論文試著去預測成熟miRNA與啟動子標靶的可能性，發現有些標靶到的位置可能重疊轉錄因子結合位置。最後，利用 pre-miRNA候選者的預測方法，成功預測出的6個pre-miRNA候選者，與hsa-mir-566高度相似（相似度為63~83 %）。
因此，有鑒於這三方面的研究，推測這些miRNAs或許在CpG islands甲基化中扮演潛在的角色，牽涉到生長發育與癌症形成的過程。未來，in vitro或in vivo實驗是必須執行去檢驗上述之預測結果。也許某些與CpG islands和啟動子區域有關的miRNAs，未來將可能跟甲基化的自體調控作用有關。

MicroRNAs ( miRNAs ) are about 22 nucleotides long small regulatory RNAs produced from hairpin-like precursors, and suppressing translation of target genes by binding to their mRNA. For this reason, miRNAs play the role of gene regulation in disease and health. Upstream regions of genes are associated with transcription, and it is known that they could have one or many transcript start sites in eukaryotes, and are named promoter regions. CpG islands are typically common near transcription start sites in vertebrates, often associated with promoter regions, and some of which are hypermethylated in cancer .
First, we focused on identifying human miRNA genes that are located near CpG island regions or promoter regions as well. It is found that a subset of miRNA genes is CpG islands-associated, in which a highly significant proportion of miRNA genes ( 74 of 462, i.e. 16.0 % ) are located within 1000 bp of a CpG island. It is also found that 10 of 462 miRNA genes are located within promoter regions, 7 of 10 miRNA genes from promoter regions are associated with CpG islands. Second, we study the possible of mature miRNAs that could target promoter regions. It is found that some of the targeted sites could overlap TF-binding sites, showing mature miRNAs maybe affect transcription of coding genes. Third, we produced a new protocol of predicting pre-miRNA candidates from intron or intergenic regions. According to our study, we have confirmed that 5 of 6 pre-miRNA candidates are associated with CpG islands, but none of them are located within promoter regions. The 6 miRNA precursor (mature) candidates are highly similar to the pre-miRNA, hsa-mir-566, which have a sequence similarity ranged from 63 to 83 % ( 79 to 89 % ).
In summary our study suggesting that these miRNAs maybe have a potential role in CpG islands methylation, involving in development and carcinogenesis. In vitro or in vivo experiments are required to perform in order to verify these candidates as new miRNA genes. Fourthermore, it is suspected that miRNA genes close to or overlapping with CpG islands and within promoter regions could be involved in autoregulation of methylation.

謝詞………………………………………………………………………i
摘要………………………………………………………………………ii
Abstract…………………………………………………………………iii
圖目錄…………………………………………………………………vii
表目錄…………………………………………………………………ix
第一章 緒論……………………………………………………………1
第一節 研究背景……………………………………………………1
一、Non-coding RNA………………………………………………1
（一）coding與non-coding………………………………………1
（二）small ncRNA………………………………………………2
二、RNA interference………………………………………………3
（一）RNAi的發現史……………………………………………3
（二）RNAi的作用機制…………………………………………4
（三）RNAi的應用………………………………………………5
三、miRNA…………………………………………………………6
（一）miRNA生源論……………………………………………6
（二）miRNA的作用機制……………………………………7 iv
四、CpG 甲基化…………………………………………………8
第二節 研究動機………………………………………………10
ㄧ、miRNA可能返回細胞核內…………………………………10
二、miRNA基因的來源位置……………………………………11
第三節 研究目的…………………………………………………12
第二章 材料與方法…………………………………………………14
第一節 人類miRNA基因在基因組中的位置……………………14
ㄧ、分析概念與流程………………………………………………14
二、核酸序列資料庫………………………………………………15
三、核酸序列排比軟體……………………………………………17
第二節 人類成熟的miRNA標靶之位置…………………………18
ㄧ、分析概念與流程………………………………………………18
二、人類成熟的miRNA標靶啟動子序列與能量計算…………19
三、人類成熟的miRNA標靶轉錄因子結合位置………………19
四、啟動子序列中可能具有的轉錄因子結合位置………………20
第三節 預測新穎的人類miRNA基因……………………………21
一、預測概念與流程………………………………………………21
二、pri-miRNA二維結構的摺疊…………………………………22
三、pre-miRNA門檻值過濾與序列裁切…………………………22 v
四、與真實pre-miRNA及其ㄧ致性區段做序列相似度排比……23
五、候選者的挑選…………………………………………………25
第三章 研究結果……………………………………………………26
第一節 人類miRNA基因在基因組中的位置……………………26
ㄧ、人類miRNA基因的鄰近基因與CpG islands………………26
二、人類miRNA基因與啟動子區域……………………………27
第二節 人類成熟的miRNA標靶之位置…………………………27
一、人類成熟的miRNA標靶啟動子序列與能量計算…………27
二、人類成熟的miRNA標靶轉錄因子結合位置………………29
三、啟動子序列中可能具有的轉錄因子結合位置………………29
第三節 預測新穎的人類miRNA基因……………………………30
第四章 結論與討論…………………………………………………32
參考文獻與網站………………………………………………………96
附錄……………………………………………………………………103
vi

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