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研究生:蒲芊蓉
研究生(外文):Pu, Cian-Rong
論文名稱:子宮內膜癌基因體與甲基化基因體的整合分析
論文名稱(外文):Integrative genomic and methylomic analysis of endometrial cancer
指導教授:賴鴻政賴鴻政引用關係
指導教授(外文):Lai, Hung-Cheng
口試委員:賴鴻政趙載光王毓淇莊樹諄
口試委員(外文):Lai, Hung-ChengChao,Tai-KuangWang, Yu-ChiChuang, Trees-Juen
口試日期:2015-05-29
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:81
中文關鍵詞:子宮內膜癌甲基化基因體
外文關鍵詞:Endometrial CancerMethylomic
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:1
表基因體的調控在癌症發展過程中扮演著重要角色。其中,部分基因會隨著癌症的發展,啟動子區的甲基化增加導致基因表現降低或靜默。因此,DNA甲基化的檢測有潛力成為癌症篩檢的方法。然而,目前沒有好的子宮內膜癌甲基化生物標記。本研究目的為分析甲基化基因體資料庫,開發子宮內膜癌的新穎甲基化生物標記。
依統計方法分析DNA甲基化基因體和轉錄基因體資料,找出候選基因,並在子宮內膜癌細胞株和抹片刷檢體進行驗證。資料庫來源:癌症基因體圖譜資料庫(TCGA)及高通量基因表達數據庫(GEO)。首先利用386位子宮內膜組織的DNA甲基化基因體資料和臨床資料,可分為成對樣本(n=33對,同病人的癌組織和非癌組織)及獨立樣本(癌組織372例,非癌組織46例),分別進行Wilcoxon 符號等級檢定與曼-惠特尼U檢定之甲基化數值差異分析,並與達統計顯著差異基因表達進行交叉比對,以篩選候選基因。利用去甲基化藥劑處理子宮內膜癌細胞株,測試細胞株的甲基化和基因表現的回復性。並驗證正常與腫瘤子宮內膜抹片刷之甲基化狀態。
DNA甲基化基因體資料經成對樣本與獨立樣本的統計分析,篩選出426個高甲基化基因和453個低甲基化基因,呈現顯著差異於子宮內膜癌組織,並整合基因表現資料,最後找出25個候選基因。其中Gene067在HEC1A及RL95-2兩株細胞在分子上的驗證符合預期,處理去甲基化藥物後,DNA甲基化降低,基因表現量增加。在抹片刷當中,顯示出Gene067和HAND2的子宮內膜癌抹片刷呈現高DNA甲基化,可以用來區分正常子宮內膜和子宮內膜癌。因此,DNA甲基化有潛力成為子宮內膜癌檢測的方法。

Epigenetics plays an important role to drive tumorigenesis. Some genes are decrease or silence by the DNA hypermethylation of promoter regions. Therefore, methylated genes potentially used to detect endometrial cancer.
We utilized DNA methylome and transcriptome data to identify candidate genes, used bioinformatics analysis of gene function prediction, and verified in endometrial cancer cell lines and tissue specimens. Database Source: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Obtained 386 DNA methylome and clinical data of endometrial tissues downloaded from TCGA data portal, and 86 transcriptomic data downloaded from GEO. The nonparametric statistics methods used to identify the differential methylation and expression of genes. The quantitative methylation-specific polymerase chain reaction (QMSP) and quantitative reverse transcription polymerase chain reaction (QRT-PCR), used to confirm the negative relation between DNA methylation and gene expression in endometrial cancer cell lines. We also confirmed the methylation level of candidate genes in subject samples with normal endometrium and endometrial cancer.
We selected the top differential methylation genes, which contain 425 high and 453 low methylation genes of cancer samples. After integrating transcriptomic data, we narrowed down 25 candidate genes. The Gene067 and HAND2 show high methylation in two endometrial cancer cell lines and endometrial cancer tissues. Thus, this study demonstrated that the gene methylation is a potential biomarker for detecting women with endometrial cancer.
總目錄

表目錄 III
圖目錄 IV
附圖目錄 VI
縮寫表 VII
中文摘要 VIII
Abstract X
第一章 緒論 1
第一節 子宮內膜癌介紹 3
第二節 子宮內膜癌的檢測及診斷工具 9
第三節 癌症與表基因體調控 11
第四節 子宮內膜與DNA甲基化 14
第二章 研究目的 16
第三章 實驗材料與方法 17
第一節 實驗流程設計 17
第二節 資料分析 18
第三節 子宮內膜細胞株驗證 21
第四章 結果與討論 27
第一節 子宮內膜組織的DNA甲基化基因體資料分析結果 27
第二節 子宮內膜組織的轉錄基因體資料分析結果 28
第三節 子宮內膜癌中基因表達與甲基化差異一致的基因 29
第四節 比較基因在子宮內膜癌細胞當中甲基化狀態及基因表達 30
第五節 比較基因HAND2在抹片刷當中,正常子宮內膜與子宮內膜樣癌的甲基化狀態 32
第六節 在比較與代謝相關基因有無之間的存活分析 33
第五章 結論 34
參考文獻 37

表目錄

表一、台灣女性每10萬人口標準化發生率 (2000年世界標準人口)2000-2011年 43
表二、子宮內膜癌的分期法(2009 FIGO) 44
表三、子宮內膜癌與甲基化相關基因 45
表四、子宮內膜癌與甲基化相關基因:討論功能但臨床證據不足 48
表五、癌症基因體圖譜資料類別 50
表六、qMSP基因引子序列 51
表七、研究樣本之人口變項 52
表八、對所有子宮內膜癌患者進行整體存活單變項Cox回歸分析 53

圖目錄

圖一、子宮內膜組織成對樣本的DNA甲基化基因體資料分析圖 54
圖二、子宮內膜組織獨立樣本的DNA甲基化基因體資料分析圖 55
圖三、子宮內膜組織中成對樣本與獨立樣本的DNA高甲基化基因體比較結果 56
圖四、子宮內膜組織中成對樣本與獨立樣本的DNA低甲基化基因體比較結果 57
圖五、子宮內膜癌轉錄基因體分析圖 58
圖六、在子宮內膜癌比對高甲基化基因及低表現基因 59
圖七、在子宮內膜癌比對低甲基化基因及高表現基因 60
圖八、GENE012基因在HEC1A細胞株中,於不同濃度去甲基化藥物中的基因和甲基化表現狀態 61
圖九、GENE041基因在HEC1A細胞株中,於不同濃度去甲基化藥物中的基因和甲基化表現狀態 62
圖十、GENE067基因在HEC1A細胞株中,於不同濃度去甲基化藥物中的基因和甲基化表現狀態 63
圖十一、GENE012基因在RL95-2細胞株中,於不同濃度去甲基化藥物中的基因和甲基化表現狀態 64
圖十二、GENE041基因在RL95-2細胞株中,於不同濃度去甲基化藥物中的基因和甲基化表現狀態 65
圖十三、GENE067基因在RL95-2細胞株中,於不同濃度去甲基化藥物中的基因和甲基化表現狀態 66
圖十四、HAND2基因在正常子宮內膜和子宮內膜樣癌抹片刷的甲基化分佈圖 67

附圖目錄

附圖一、2000-2011年台灣女性子宮內膜癌發生率 68
附圖二、子宮內膜月經週期變化 69
附圖三、表基因體調控 70
附圖四、人類甲基化450K芯片原理 71

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