人類與老鼠microRNAs之預測、驗證、與功能性分析

Abstract

重要性和研究動機: MicroRNA (簡稱miRNAs)是一種non-coding 基 因，可以與mRNA 的片段(sites)進行Hybridization，進而影響到基因的表 現 (Gene expression)。目前生物體中miRNA 的功能，尚未被研究的很清 楚。因此，我們認為快速而有效率的miRNA 電腦分析工具是迫切需要的。 而對於基因體來說，建立miRNA 基因的位置和miRNA 目標的基因之間的對 應關係，對於基因體的研究，例如基因調控、替代性剪接、及後轉錄調控， 會有很大的幫助。 研究目的: (1) 發展一個整合性資料庫, miRNAMap, 以儲存已知的 miRNA 基因, 預測的 miRNA基因, 已知的 miRNA target, 以及人類和老 鼠中的預測的miRNA targets。 (2) 用分子生物實驗來確認我們所預測 miRNAs。 (3)我們希望運用array-CGH資料和微陣列基因表現資料來探討 miRNA和miRNA target之間的調控關係。我們也希望能夠有系統地偵測出與 疾病/癌相關的miRNAs。 主要研究方法: (1) 我們從miRBase資料庫得到人、老鼠、大鼠和狗的 miRNAs，並且運用文獻考察, 提取經實驗證實的 miRNA targets。 miRNA 先驅物將被預測出來。我們將運用 miRanda 在人類和老鼠中基因的 3'-UTR中的保守序列區中, 找尋miRNA targets。(2) 同時保守(Conserved) 於在人類和老鼠中miRNA candidates, 將由Northern Blotting來實驗證 實。 (3) 基於建立的 miRNAMap 資料庫和實驗證實的miRNAs, 我們有系統 地參考array-CGH 的資料及微陣列基因表現資料, 來找出跟疾病和癌症有 關的 miRNAs。 預期研究成果: (1) 我們將成功建立miRNAMap 資料庫。它將提供全面 性的 miRNA 資訊, 已知的 miRNAs, 跨物種的比較, 基因註解和對其他生 物資料庫橫向聯結。我們並將提供原文模式和圖形化的網路界面以有利於 miRNAMap資料的檢索。(2) 我們希望超過三十個以上的miRNAs, 能夠在人 類或老鼠的實驗中被確認, 至少15個新的miRNAs能夠被發現及確認。 (3) 我們期望能夠發展一個系統化的分析方法來找出和疾病和癌症相關連的 miRNAs。 這個研究計畫的主要貢獻如下。(1) 為了建立microRNA資料庫以儲存 已知的 miRNAs , 新發現的 miRNAs 和預測的 miRNAs 。 (2) 我們期望許 多新的 miRNAs 在這個研究中被經實驗證實。 (3) 跟疾病和癌有關的 miRNAs, 能夠有系統地被分析, 以找出跟疾病和癌有關的microRNA 調控 機制。

Significance and Motivation: Recent work has demonstrated that microRNAs (miRNAs) are involved in critical biological processes by suppressing the translation of coding genes. The specific functions of the miRNAs are unknown in most eukaryotic genomes. Therefore, computational methods for efficiently identifying the miRNA precursors, mature miRNAs and their targets in a genome are crucial. The comprehensive mapping between all the known/putative microRNA genes and their target mRNAs in genomes will also be very effective for further investigation of gene regulation, post-transcriptional control, alternative splicing pathway and other mechanisms in biological systems. The Specific Aims: (1) To develop an integrated database, miRNAMap, to store the known miRNA genes, the putative miRNA genes, the known miRNA targets and the putative miRNA targets in human and mice. (2) To experimentally confirm the predicted miRNAs, which are both conserved in human and mice. (3) To investigate the regulatory relationships between miRNAs and their targets by using array CGH data and microarray expression profiles. To systematically identify disease/cancer-related miRNAs. The Proposed Methods: (1) The known miRNA genes in four mammalian genomes such as human, mouse, rat and dog will be obtained from miRBase, and experimentally validated miRNA targets will be extracted in a survey of the literature. Putative miRNA precursors will be computationally identified using a non-coding RNA prediction tool based on comparative sequence analysis. The mature miRNA of the putative miRNA genes will be determined using a machine learning approach. miRanda will be applied to predict the miRNA targets within the conserved regions in 3』-UTR of the genes in the human and mouse. (2) The miRNA candidates conserved in human and mouse will be identified and experimentally verified by Northern Blotting. Several human cell-lines and mouse tissues are selected for the experiments for monitoring the tissue-specificity of the miRNAs. (3) Based on the constructed miRNAMap database and experimental validated results, the disease/cancer-related miRNAs can be systematically analyzed by referring to array-CGH data and microarray gene expression data. The Anticipated Results: (1) The miRNAMap database will be constructed. It will provide comprehensive miRNA information, the expression profiles of the known miRNAs, cross-species comparisons, gene annotations and cross-links to other biological databases. Both textual and graphical web interface will be provided to facilitate the retrieval of data from the miRNAMap. (2) We hope more than thirty miRNA candidates can be confirmed experimentally and at least 15 novel miRNAs in human or mouse can be identified. (3) We expect that a systematic approach can be developed to investigate the regulatory relationships among miRNAs, miRNA target genes and to identify disease/cancer-related miRNAs. The main contributions of this project are addressed as follows. (1) To establish a microRNA databases to curate known miRNAs, novel miRNAs and predicted miRNAs. (2) A variety of novel miRNAs are expected to be identified in this investigation. (3) Disease/cancer-related miRNAs can be determined to effectively enhance the investigation of the disease-cause mechanisms involved by microRNAs.