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系統識別號 U0026-2208201617470900
論文名稱(中文) Zinc Finger Protein 496 在肝癌中是一個有潛力的 Wnt/β-catenin 路徑中的標的基因
論文名稱(英文) Zinc Finger Protein 496 is a potential target gene of Wnt/β- catenin pathway in hepatocellular carcinoma
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 104
學期 2
出版年 105
研究生(中文) 張瀞云
研究生(英文) Ching-Yun Chang
學號 T36034115
學位類別 碩士
語文別 中文
論文頁數 55頁
口試委員 指導教授-何中良
口試委員-楊孔嘉
口試委員-黃溫雅
口試委員-蔡弘文
中文關鍵字 Wnt/β-catenin傳遞路徑  生物資訊分析  ZNF496 
英文關鍵字 Wnt/β-catenin pathway  Bioinformatics  ZNF496 
學科別分類
中文摘要 腫瘤胚胎基因在胚胎或胎兒中表現,而正常成人組織中表現量低或是不表現,但腫瘤組織中又再度表現。Wnt/β-catenin 傳遞路徑在胚胎發育、細胞增生、細胞分化、細胞再生、細胞自我更新及腫瘤生成過程中扮演著重要的角色。先前研究都指出異常活化此路徑及其下游的標靶基因會造成許多癌症,其中包含了肝癌。肝癌是全球最常見的惡性腫瘤之ㄧ,也是第四大癌症導致死亡。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 等。因此,透過鑑定Wnt/β-catenin 標靶基因去了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Biodatabase運用來尋找有潛力的 Wnt/β-catenin 標靶基因,其中一個為Zinc Finger Protein 496 (ZNF496) 。所以本篇研究主要是在探討ZNF496 是否是Wnt/β-catenin signaling pathway 的target gene,另外也探討ZNF496 在肝癌中所扮演的角色及功能。首先利用 Lithium chloride 和 Wnt-3A 條件培養液去活化Wnt/β-catenin 傳遞路徑,再經由半定量 RT-PCR 發現 ZNF496 的 mRNA 表現量會增加。進一步利用染色質免疫沉澱法 (ChIP) 、螢光素酶偵測法 (luciferase assays) 、突變法 (mutation assays )證實 β-catenin/TCF complex 會結合於 ZNF496 的 promoter 位置。結果顯示ZNF496為有潛力是Wnt/β-catenin 的target gene。除此之外,我們發現 ZNF496 會在肝癌腫
瘤組織大量表現。而overexpression 實驗結果顯示為 ZNF496 會促進幹細胞特性、上皮-間質轉化 (EMT) 及 細胞增生,而使用 XAV939 (Wnt/β-catenin 傳遞路徑的抑制劑) 可以抑制ZNF496 的表現。
英文摘要 Misregulation of the Wnt/β-catenin signaling pathway occurred in various human cancers including liver cancer. In our laboratory, we developed a combined bioinformatics and experimental approach to find a potential target gene of the Wnt/β-catenin signaling pathway. One of these candidate genes is Zinc Finger Protein 496 (ZNF496). ChIP assay, luciferase assay and mutation assay suggested that the TCF binding site of ZNF496 promoter was critical for β-catenin /TCF dependent regulation. In this study, we investigate the function and characteristics of ZNF496 in human HCC cell lines. The results showed that overexpression of ZNF496 could promote stemness, EMT and cell proliferation. XAV939 which is the Wnt/β-catenin specific inhibitor could inhibit the expression of the target genes of Wnt/β-catenin, such as c-myc, cyclin D1 and ZNF496. These results indicate that ZNF496 may be a potential target gene of Wnt/β-catenin pathway and may play important roles in HCC.
論文目次 摘要....................................................I
英文延伸摘要............................................II
致謝..................................................VII
目錄.................................................VIII
表目錄.................................................XI
圖目錄................................................XII
附錄.................................................XIII
第一章 緒論..............................................1
1.1 Wnt傳遞路徑的總覽....................................1
1.2 Wnt/β-catenin傳遞路徑與肝臟之相關性...................2
1.2.1 Wnt/β-catenin傳遞路徑與肝臟生成之相關性..............2
1.2.2 Wnt/β-catenin傳遞路徑與肝癌生成之相關性..............2
1.3 目前 Wnt/β-catenin標靶基因在臨床之應用.................3
1.4 利用生物資訊方法尋找Wnt/β-catenin標靶基因..............3
1.4.1 生物資料庫 (Bio-database)的建立.....................3
1.4.2 利用生物資訊方法尋找新穎的Wnt/β-catenin標靶基因.......4
1.5 ZNF496可能為Wnt/β-catenin傳遞路徑標靶基因.............5
1.5.1 利用Lithium chloride活化Wnt/β-catenin傳遞路徑並觀察ZNF496基因之mRNA表現.....................................5
1.5.2 利用Wnt-3A條件培養液活化Wnt/β-catenin傳遞路徑並觀察ZNF496基因之mRNA表現.....................................6
1.5.3 以染色質免疫沉澱法觀察ZNF496是否與β-catenin/TCF complex有交互作用........................................7
1.5.4 利用Luciferase assay分析β-catenin是否調節ZNF496 promoter................................................7
1.5.5 ZNF496在肝癌組織中的表現............................8
1.6 實驗目的…............................................8
第二章 實驗材料與方法.....................................9
2.1 細胞培養.............................................9
2.2 RNA萃取 (RNA extraction)...........................11
2.3 反轉錄聚合酵素連鎖反應...............................11
2.4 DNA 純化...........................................12
2.5 核酸接合作用 (DNA Ligation).........................13
2.6 限制酶切割DNA實驗...................................13
2.7 勝任細胞製備........................................13
2.8 轉型作用 (transformation)...........................14
2.9 質體抽取 (plasmid DNA extraction)...................14
2.10 反轉錄病毒製備.....................................15
2.11 轉染作用 (transfection)............................16
2.12 細胞相關實驗.......................................17
2.13 西方墨點法.........................................17
2.14 統計分析...........................................22
第三章 實驗結果.........................................23
3.1觀察ZNF496在不同肝癌細胞中的表現.......................23
3.2建構穩定表達ZNF496之肝癌細胞株.........................23
3.3 觀察過表達ZNF496 的肝癌細胞是否會增加癌細胞中幹細胞之特性.....................................................23
3.4 探討過表達ZNF496與上皮間質轉化之分子機轉...............24
3.5 探討過表達ZNF496與細胞增生之相關性....................25
3.6 利用XAV939抑制Wnt/β-catenin傳遞路徑探討ZNF496與Wnt/β-catenin傳遞路徑之相關性..................................25
3.7 利用cBioPortal for Cancer Genomics database 分析The Cancer Genome Atlas(TCGA)中ZNF496與肝癌之相關性..........26
第四章 討論.............................................27
第五章 參考文獻.........................................30
第六章 表...............................................35
第七章 圖...............................................38
第八章 附錄.............................................47
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