Dissecting Epigenetic Alterations between Hepatitis B Virus and Hepatitis C Virus-infected Hepatocellular Carcinoma using CpG Island Microarray

肝細胞癌(簡稱肝癌)的發生率在全球惡性腫瘤中排名第六且預後差，其死亡率是所有癌症中的第三位。肝癌的發生是多因素且多步驟的進程，不但病因複雜且臨床症狀不易顯現，目前已知全球肝癌發生之主要危險因子為B型肝炎病毒(HBV)和C型肝炎病毒(HCV)的感染，並經証實超過80%的肝癌與HBV和HCV感染有關且能使致癌率提升20倍。許多肝癌發生的研究報導顯示，遺傳事件與上遺傳事件（如DNA的甲基化）等基因體的改變，與基因的表現異常進而影響細胞轉型成惡性腫瘤的現象有關。本研究從台北榮民總醫院所提供的153位肝癌病患中，根據其臨床病理資料挑選出10個肝癌病患的檢體，利用甲基化晶片(DMH)技術進行基因體全面性偵測肝腫瘤組織的甲基化程度差異。根據初步晶片分析結果，肝腫瘤組織在HBV與HCV不同的病毒感染情況下，從約12000個基因中經篩選出有甲基化差異的總共95個，其中有較高基因甲基化程度的71個基因屬於HBV感染相關的檢體，另外24個基因則相反的在HCV感染相關的檢體中具有較高的基因甲基化程度。另外本研究也利用MS-PCR技術，針對不同病毒感染而有基因甲基化差異的基因群，進行單一基因甲基化程度的驗證。分析153位肝癌病患中Wwox、p16、GSTP1、DAPK、hMLH與MGMT等基因的結果中發現，其甲基化比例分別為39.87％、46.41% 、33.99%、39.22%、3.7%、與0%。上述的結果顯示MS-PCR之驗證與晶片呈現趨勢大致相同，其中Wwox基因為HBV相關之高度甲基化的基因，p16基因為HCV相關之高度甲基化的基因，GSTP1與DAPK 基因則為全面性的高度甲基化基因，hMLH以及MGMT是屬於沒有甲基化的基因。進一步針對8個肝癌細胞株裡的DAPK基因進行甲基化研究，在其5’調節區的CpG小島片段中設計了一段引子對並進行偵測，發現有三個細胞株具有高度甲基化情形，再針對細胞株HA22T/VGH進行5’-Aza-dC/TSA去甲基化試驗，證實DAPK基因去甲基後能使基因表現有明顯回復，這亦是第一次發現DAPK基因甲基化與基因表現降低相關。另外，根據晶片叢集分析發現基因甲基化的高低差異與其臨床病理有顯著相關，其基因甲基化愈高的檢體樣本不但發病年齡低且其腫瘤相對也較大。本研究希望藉由甲基化晶片分析獲得大量肝癌相關之異常甲基化基因群，並以進一步的分析試驗其甲基化意義，期能描繪出甲基化於肝癌發生所扮演的機制藍圖。Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies and a major cause of cancer-related deaths worldwide. Most HCCs exhibit characteristics compatible with chronic hepatitis and cirrhosis caused by persistent infection of hepatitis B virus (HBV) and/or hepatitis C virus (HCV). However, the molecular mechanisms of hepatocarcinogenesis associated with hepatitis viral infection have not been clarified. It is now clear that aberrant DNA methylation observed in cancer cells and also involved in tumor development. In order to investigate further this widespread epigenetic event in the tumor genome, we have applied an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in liver cancer. DMH microarrays containing about 12,000 CpG islands and subsequent selection yielded 95 clones for which methylation status differed between the two types of virus-infected HCCs. The methylation statuses of 74 of these 95 genes were serious in HBV-associated HCCs, and the remaining 21 hypermethylated genes were found in HCV-associated HCCs. In this study, we selected 6 candidate genes to validate their promoter region methylation status in 153 HCC tissues detecting by methylation-specific PCR (MS-PCR). Using this candidate gene approach, we showed that Wwox(39.87％) p16 (46.41%), GSTP1 (33.99%), DAPK (39.22%), hMLH (3.7%) and MGMT(0％) were have diverse methylation frequency in HCCs. The results from microarray and MS-PCR analysis were almost consistence. Wwox gene was the hypermethylated gene correlated with HBV-associated HCCs, and the p16 gene gene was the hypermethylated gene correlated with HCV-associated HCCs, and DAPK and GSTP1 genes were the universal hypermethylated genes in our examined HCCs. Besides, hMLH and MGMT gene were showed no methylation in HCCs. Furthermore, we defined a CpG-rich region of 5' regulatory region of DAPK gene and examined the methylation status in 8 hepatoma cell lines by MS-PCR. The demethylation assay was applied to investigate the relationship between methylation of DAPK and its mRNA transcription by treatment with 5-aza-2'deoxycytidine (5'-Aza-dC) and trichostatin A (TSA). Our data showed that the restoration of DAPK expression in heavily methylated hepatoma cell line (HA22T/VGH) was quite obvious after 4 days of 5'-Aza-dC treated. Our results suggested that the epigenetic control in DNA methylation level is an important mechanism for DAPK gene regulation. Moreover, we also discover another thrilling hierarchical clustering result from DMH microarray, which was the significant correlation between hypermethylated gene group and the relative younger age and larger tumor size. This early finding lays the groundwork for DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in HCCs.摘要(中文)............................................................................................................. i 摘要(英文).............................................................................................................ii 表次.......................................................................................................................iv 圖次........................................................................................................................v 壹、 緒言..............................................................................................................1 貳、 文獻檢討........................................................................................................2 一、肝癌 — 常見惡性腫瘤 （一） 肝癌發生成因簡介.........................................................................2 （二） 肝癌與甲基化.................................................................................6 二、基因甲基化的偵測技術分析 （一） 細胞總量甲基之偵測.....................................................................7 （二） 單一基因CpG位置甲基化檢測....................................................9 （三） 多基因之甲基化程度分析..........................................................11 （四） 基因體全面性甲基化分析..........................................................13 三、甲基化與腫瘤抑制基因 （一） 生物體內的甲基化機制...............................................................14 （二） 特殊腫瘤抑制基因探討...............................................................18 四、甲基化之於肝癌研究 — 啟發與展望..................................................21 參、 材料與方法.............................................................................................23 ㄧ、組織樣本與細胞株來源與保存 （一） 肝腫瘤組織樣本來源與保存.......................................................23 （二） 肝癌細胞株來源與保存...............................................................23 二、差異性甲基化雜合晶片(Differentiation methylation hybridization, DMH) （一） 樣本製備.......................................................................................24 （二） 螢光標定與雜合反應...................................................................27 （三） 螢光偵測與分析...........................................................................29 三、甲基特異性聚合酶連鎖反應(Methylation-specific PCR, MS-PCR) （一） DNA之亞硫酸鈉化學修飾.........................................................29 （二） MS-PCR分析...............................................................................29 四、 mRNA表現分析 （一）萃取肝臟腫瘤組織與肝癌細胞株RNA........................................30 （二）反轉錄聚合酶連鎖反應(RT-PCR).................................................32 五、 肝癌細胞株去甲基藥物試驗 （一）去甲基藥物(5-Aza-dC)處理..........................................................32 （二）COBRA(combined bisulfite restriction analysis)技術分析 DAPK基因去甲基化處理效應....................................................33 （三）甲基化特異性核酸定序分析(methylation-specific sequencing; MS-sequencing)............................................................................34 肆、 結果與討論.............................................................................................36 ㄧ、 以差異性甲基化雜合晶片(DMH)分析肝臟腫瘤組織樣本 （一）差異性甲基化雜合晶片分析.........................................................36 （二）晶片總分析結果分群探討與不同病毒感染形式之關聯性.........41 1. HBV病毒感染之肝癌組織具有高度甲基化的基因群........41 2. HCV病毒感染之肝癌組織具有高度甲基化的基因群........43 3. DMH晶片分析呈現全面性高度甲基化(universal hypermethylation)的基因群..................................................44 （三）晶片叢集分析結果與臨床資料綜合討論.....................................45 二、 甲基化特異性PCR技術分析肝臟腫瘤組織樣本於五個腫瘤抑 制基因之甲基化狀態 (一) WWOX、p16、GSTP1、DAPK、MGMT、hMLH基因於 肝臟腫瘤組織之甲基化狀態........................................................50 (二) p16、GSTP1、DAPK基因於肝臟腫瘤組織之甲基化 狀態與臨床資料分析討論............................................................59 (三) WWOX、p16、GSTP1、DAPK、MGMT、hMLH基因於 MS-PCR技術分析與DMH甲基化分析結果之綜合討論..........63 三、偵測DAPK基因於8株人類肝癌細胞株之甲基化狀態與mRNA 表現情形 (一) DAPK基因於8株人類肝癌細胞株之甲基化狀態...................67 (二) DAPK基因於8株人類肝癌細胞株之mRNA表現情形..........67 四、肝癌細胞株HA22T/VGH之去甲基化試驗 (一) 以MS-PCR與COBRA技術驗證HA22T/VGH細胞株去 甲基化試驗之有效性....................................................................70 (二) HA22T/VGH細胞株去甲基化處理後之DAPK mRNA 表現量增加....................................................................................71 (三) DAPK基因5’端調節區之轉錄因子結合位置預測分析與 甲基化特異性定序分析................................................................71 五、肝癌病人檢體之DAPK mRNA表現偵測.............................................72 伍、 結論...........................................................................................................76 陸、 參考文獻...................................................................................................7

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