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系統識別號 U0026-2307201114214300
論文名稱(中文) 結合生物資訊暨實驗篩選以尋找 Wnt/β-catenin 傳遞路徑之新穎基因
論文名稱(英文) Identifying novel target genes of the Wnt/β-catenin signaling pathway using a combined bioinformatics and experimental approach
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 99
學期 2
出版年 100
研究生(中文) 陳怡文
研究生(英文) Yi-Wen Chen
學號 T36984093
學位類別 碩士
語文別 中文
論文頁數 78頁
口試委員 指導教授-何中良
口試委員-徐麗君
口試委員-楊順發
中文關鍵字 Wnt/β-catenin 傳遞路徑  標靶基因  Expressed Sequence Tags 資料庫  生物資訊分析  β-catenin/TCF complex  染色質免疫沉澱法 
英文關鍵字 Wnt/β-catenin signaling pathway  target genes  Expressed Sequence Tags  bioinformatics  β-catenin/TCF complex  chromatin immunoprecipitation assay 
學科別分類
中文摘要 Wnt/β-catenin 傳遞路徑在胚胎發育及腫瘤生成過程中都扮演著相當重要的角色。在許多人類的惡性腫瘤中皆會發現不正常活化此路徑及其下游的標靶基因,能促進腫瘤的生成。目前已有多個 Wnt/β-catenin 標靶基因被發現在腫瘤生成中扮演關鍵角色,如 c-myc,cyclin D1,MMPs 及survivin 等。因此,透過鑑定 Wnt/β-catenin 標靶基因去瞭解其所調控的腫瘤生成是很重要的議題。在過去實驗室建立了一個結構完整、具擴展性的生物資料庫,簡稱 Bio-database。我們將此生物資料庫運用來尋找有潛力的 Wnt/β-catenin 標靶基因。用已知的 65 個 Wnt/β-catenin 基因設定參數後,從資料庫中初步找出 161 個基因,並經由文獻搜尋將其分成 111 個已知功能基因和 50 個未知功能基因。而在已知功能基因中確實存在高比例的 Wnt/β-catenin 標靶基因,所以我們相信在 50 個未知功能基因中應該也存在著新穎的 Wnt/β-catenin 標靶基因。我們從 50 個基因中隨機挑選 25 個基因去鑑定其是否為 Wnt/β-catenin 標靶基因。利用 Lithium chloride 和 Wnt-3A 條件培養液,兩種不同的方式去活化 SK-hep-1 細胞的 Wnt/β-catenin 傳遞路徑,再經由半定量 RT-PCR 發現 25 個未知基因中,有 4 個基因 (U496、U75、U39 及 FLJ10156) 的 mRNA 表現量會增加。進一步去探討這 4 個基因的表現是否直接被 β-catenin/TCF complex 所調控,透過染色質免疫沉澱法證實 β-catenin/TCF complex 會結合於 U496、U75 及 U39 的 promoter 位置。除此之外,我們發現這三個基因皆會在肝癌腫瘤組織大量表現。最後去評估這三個基因在腫瘤大量表現的臨床意義,將基因表現情形與肝癌病理參數進行相關性分析,但初步發現這些基因與肝癌病理特質沒有統計上的意義。總結目前的實驗結果,我們利用生物資訊分析結合實驗篩選可找到 U496、U75 及 U39 為 Wnt/β-catenin 的標靶基因,並且在腫瘤組織中會大量表現,推測在腫瘤生成過程中應有其特殊的生物意義。
英文摘要 The Wnt/β-catenin signaling pathway plays an important role in embryogenesis and tumorigenesis. Misregulation of the Wnt/β-catenin signaling pathway and aberrant activation of target genes of Wnt/β-catenin are common in various human cancers and contribute to cancer progression. Several target genes are known to be key players in tumourigenesis, such as c-myc, cyclin D1, MMPs or survivin. Therefore, identifying the target genes of Wnt/β-catenin signaling pathway is important for understanding Wnt/β-catenin-mediated carcinogenesis. Previously, our lab set up a structured and scalable bioinformatics database- Bio-database. We applied Bio-database to search candidate target genes of Wnt/β-catenin. Using 65 published genes of Wnt/β-catenin as references to set the thresholds of bioinformatics analysis yielded 111 genes with known functions and 50 genes with unknown functions. In the 111 known genes, we indeed found a high percentage of target genes of Wnt/β-catenin. Hence, we believed that the 50 unknown genes may contain novel target genes of Wnt/β-catenin. We chose 25 of 50 unknown genes to examine whether these canadidate genes were novel target genes of Wnt/β-catenin. SK-hep-1 cells were treated with Lithium chloride or Wnt3A-conditioned medium, two different methods to activate the Wnt/β-catenin signaling pathway. By semi-quantitative RT-PCR, the mRNA expression of four genes (U496, U75, U39 and FLJ10156) was found to be up-regulated in cells with activated Wnt/β-catenin pathway. Chromatin immunoprecipitation (ChIP) assay was performed to further examine whether the expression of four genes was directly regulated by β-catenin /TCF (T cell factor) complex. The ChIP assay demonstrated direct interaction of β-catenin/TCF complex with the promoter of three genes (U496, U75 and U39). In addition, the mRNA expression of the three genes was significantly up-regulated in some of the hepatic tumors. To assess the potential clinical significance of the three genes, we examined their correlation with a variety of pathological factors. However, no statistical significance was observed between the three genes and pathologic features in liver cancer. In conclusion, the U496, U75 and U39 genes are direct targets of Wnt/β-catenin and are overexpressed in hepatic tumor. They may play an important role in tumorigenesis.

論文目次 摘 要................................................I
Abstract............................................III
致 謝................................................V
目 錄................................................VI
表 目 錄.............................................IX
圖 目 錄..............................................X
第一章 緒論........................................1
1.1 Wnt 傳遞路徑的總覽...........................1
1.1.1 Wnt 家族........................................1
1.1.2 Wnt 受體........................................2
1.1.3 細胞內訊息傳遞路徑................................4
1.2 Wnt/β-catenin 傳遞路徑與癌症形成之關係...............5
1.2.1 Wnt/β-catenin 傳遞路徑之調控機制..................5
1.2.2 Wnt/β-catenin 傳遞路徑與癌症生成之相關性...........7
1.2.3 β-catenin、APC 及 Axin 的基因突變位...............7
1.3 目前 Wnt/β-catenin 標靶基因在臨床之應用..............9
1.3.1 Wnt/β-catenin 標靶基因...........................9
1.3.2 臨床應用.........................................10
1.4 TCF/LEF 家族......................................11
1.5 利用生物資訊方法尋找 Wnt/β-catenin 標靶基因..........12
1.5.1 表現序列標幟 (Expressed Sequence Tags , EST).....12
1.5.2 生物資料庫 (Bio-database) 的建立..................13
1.5.3 利用生物資訊方法尋找新穎的 Wnt/β-catenin 標靶基因...15
1.6 實驗目的...............................................16
第二章 實驗材料與方法....................................18
2.1 細胞的培養程序......................................18
2.2 細胞相關實驗........................................20
2.3 西方墨點法..........................................21
2.4 抽取 total RNA.....................................26
2.5 反轉錄聚合酵素連鎖反應.........................28
2.6 染色質免疫沉澱法.................................30
2.7 DNA 純化.......................................33
2.8 生物資訊軟體....................................33
2.9 統計分析........................................34
第三章 實驗結果.........................................35
3.1 利用 Lithium chloride 誘導 Wnt/β-catenin 傳遞路徑之活化並觀
察 Wnt/β-catenin 候選基因之 mRNA 表現......................35
3.2 利用 Wnt-3A 條件培養液誘導 Wnt/β-catenin 傳遞路徑之活化並觀察 Wnt/β-catenin 候選基因之 mRNA 表現......................36
3.3 建立染色質免疫沉澱法.................................38
3.4 探討 β-catenin/TCF complex 對 U496 promoter 區域之結合..40
3.5 探討 β-catenin/TCF complex 對 U75 promoter 區域之結合...41
3.6 探討 β-catenin/TCF complex 對 U39 promoter 區域之結合...42
3.7 探討 β-catenin/TCF complex 對 FLJ10156 promoter 區域之結合........................................................42
3.8 基因在肝癌腫瘤組織中的表現及其臨床意義....................43
第四章 討論....................................45
第五章 參考文獻.............................50
第六章 表......................................55
第七章 圖......................................66
作 者 自 述....................................78
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