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研究生: 郭禹昊
Kuo, Yu-Hao
論文名稱: 驗證潛在Wnt標靶基因的TCF結合位點
Validation of TCF binding sites of potential Wnt direct target genes
指導教授: 何中良
Ho, Chung-Liang
學位類別: 碩士
Master
系所名稱: 醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 94
中文關鍵詞: Wnt/β-catenin信號傳遞路徑(Wnt/β-catenin signaling pathway)癌症(Cancer)Wnt響應元件(Wnt response element, WRE)TCF結合位點(TCF-binding element, TBE)TBE預測工具(TBE prediction tool)
外文關鍵詞: Wnt/β-catenin signaling pathway, Cancer, Wnt response element (WRE), TCF-binding element (TBE), TBE prediction tool
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  • 中文摘要 I 英文延伸摘要 (Extended Abstract) III 誌謝 IX 目錄 XI 表目錄 XV 圖目錄 XVI 附錄目錄 XVII 縮寫字表 XVIII 第一章 緒論 1 1.1 肝癌 1 1.1.1 簡介 1 1.1.2 肝癌分期 1 1.1.3 臨床診斷及治療 3 1.2 Wnt傳遞路徑 6 1.2.1 Wnt蛋白配體 (Wnt Ligand) 6 1.2.2 Wnt受體 (Wnt Receptor) 7 1.2.3 Wnt傳遞路徑 (Wnt pathway) 9 1.3 Wnt/β-catenin訊號傳遞路徑於臨床之影響 12 1.3.1 Wnt/β-catenin與癌症 12 1.3.2 Wnt/β-catenin於臨床之應用 13 1.4 生物資訊 16 1.4.1 目標轉錄因子TCF7L2 16 1.4.2 PROMO & JASPAR 16 1.4.3 ChIP-ATLAS 17 1.5 實驗室先前研究 17 1.5.1 建立生物資料庫 (Bio-database) 17 1.5.2 尋找Wnt/β-catenin傳遞路徑之新穎基因 18 1.6 候選基因POMGNT2、METTL1、ETV4、CCDC77和GPR107之簡介 19 1.6.1 ETV4 20 1.6.2 POMGNT2 20 1.6.3 METTL1 20 1.6.4 CCDC77 21 1.6.5 GPR107 21 1.7 實驗目的 21 第二章 實驗材料與方法 23 2.1 生物資訊軟體 (Bioinformatics Analysis) 23 2.1.1 利用NCBI和GeneCards數據庫查詢基因資料 23 2.1.2 利用the Wnt homepage數據庫查詢和比較基因資料 23 2.1.3 利用Ensembl找尋目標基因的Promoter 23 2.1.4 利用NEBcloner和Cleavage Close to the End of DNA Fragments分析限制酶特性 24 2.1.5 利用JASPAR預測轉錄因子的結合位點 24 2.1.6 利用ChIP-Atlas查詢轉錄因子的結合位點 24 2.1.7 利用NCBI primer-blast設計引子 25 2.1.8 利用QuickChange Primer Design設計點突變引子 25 2.1.9 利用NCBI BLAST進行序列比對 25 2.1.10 利用NCBI SNP和Taiwan BioBank分析SNP 25 2.2 分子生物技術 (Molecular Biotechnology) 26 2.2.1 聚合酶鏈鎖反應 (Polymerase Chain Reaction) 26 2.2.2 點突變PCR (Site Directed Mutagenesis) 28 2.2.3 洋菜瓊脂製備 (Agarose Gel) 30 2.2.4 DNA純化 (DNA Purification) 31 2.2.5 質體構築 (Construct) 32 2.2.6 質體DNA提取 (Plasmid DNA Extraction) 35 2.2.7 本實驗基因之質體構築 (Target Gene Construction) 38 2.3 細胞培養與實驗 (Cell Culture and Experiments) 39 2.3.1 實驗細胞株 (Cell Lines) 39 2.3.2 細胞溶液 (Cell Solution) 39 2.3.3 細胞解凍 (Thawing Cells) 40 2.3.4 細胞冷凍 (Freezing Cells) 40 2.3.5 細胞培養/繼代培養 (Cell culture/Subculture) 40 2.3.6 細胞計數 (Counting Cells) 41 2.3.7 細胞轉染 (Transfection) 41 2.3.8 雙冷光素酶報告基因檢測 (Dual-Luciferase Reporter Assay) 42 2.4 數據分析與統計 (Statistical Analysis) 43 第三章 實驗結果 44 3.1 不同資料庫之比較 44 3.2 候選基因之Promoter構築與點突變 46 3.2.1 ETV4位點突變 46 3.2.2 POMGNT2位點突變 47 3.2.3 CCDC77質體構築 47 3.2.4 GPR107質體構築 48 3.3 驗證候選基因是否為Wnt/β-catenin之標靶基因 49 3.3.1 建立Luciferase Reporter Assay流程 49 3.3.2 ETV4 promoter活性 50 3.3.3 POMGNT2 promoter活性 51 3.3.4 CCDC77 promoter活性 51 3.3.5 GPR107 promoter活性 52 第四章 討論 53 4.1 探討ChIP-seq和Position Weight Matrices 53 4.2 探討各目標基因 54 第五章 表 56 第六章 圖 62 第七章 參考文獻 74 第八章 附錄 81

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