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Dissecting Epigenetic Alterations between Hepatitis B Virus and Hepatitis C Virus-infected Hepatocellular Carcinoma using CpG Island Microarray
Differential methylation hybridization (DMH)
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|摘要:||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.
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