慢性B型肝炎到肝細胞癌進程之甲基化變動：試驗性研究

Dynamic change of DNA methylation during progression from chronic HBV carrier state to Hepatocellular carcinoma: A pilot study

高惠娟(Hui-Chuan Kao)

指導教授 ： 于明暉

國立臺灣大學/公共衛生學院/流行病學與預防醫學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602260

摘要

研究背景與目的： DNA甲基化是表觀遺傳學的機制之一，在調節基因表現量上扮演重要的角色且已被證實與癌症發生有密切的相關。透過先前白血球全基因體甲基化分析，找出一群可能可以用於預測肝細胞癌的甲基標記。本篇研究目的欲觀察從慢性B型肝炎發展成肝細胞癌進程中，DNA甲基化的變動情形以及探討肝細胞癌危險因子暴露與否，是否會影響甲基化的變動。

研究方法：利用焦磷酸定序(pyrosequencing)定量5支探針所涵蓋CpG位點的甲基化程度，使用48位發生肝細胞癌年齡小於50歲的患者，距確診前0-13年間所提供的周邊白血球DNA檢體(每個患者提供的次數範圍為1-6次，一共有126個血液檢體)作為檢測，並以48名非肝細胞癌患者作為對照組。依照距離確診前5個時間區間，以廣義線性模式檢測甲基化程度與肝細胞癌之間的關係。再者，使用ROC曲線下面積評估隨著距離確診時間的遠近，選用的甲基標記，其預測能力是否有改變。最後，以線性混合效果模型探討已知肝細胞癌危險因子與甲基化變動之間的關連性。

研究結果：於此篇研究選擇的甲基標記，其甲基化程度不但與肝細胞癌之間具相關性並且在確診前5個時間分群之間，甲基化程度維持穩定；再者，選用的甲基標記，其AUC不隨時間有所改變。但在另一方面，15名肝細胞癌患者距確診時間3年以內的甲基化程度，越接近確診時間，甲基化程度越高。最後，e抗原與吸菸，兩個肝細胞癌危險因子會影響甲基化的變動情形。

結論：從慢性B型肝炎到肝細胞癌的進程，所選用的甲基標記，其變動情形在個體之間並不一致，以至於整體變動情形並不明顯。此外，已知的肝細胞癌危險因子可能會影響甲基化的變動。

關鍵字

乙型肝炎 ； 肝細胞癌 ； 甲基化 ； 變動

並列摘要

Background and Aims： DNA methylation plays a pivotal role in the regulation of gene expression and aberrant methylation has been shown to be involved in a spectrum of carcinogenic process. Through genome-wide scan on leukocyte DNA samples, a set of DNA methylated genes have been identified as potential biomarkers for predicting hepatocellular carcinoma. The specific aims of this study were to evaluate the dynamics of selected candidate methylation markers during transition from asymptomatic HBV carrier state to HCC and to find out whether this dynamic change depended on exposure to putative HCC risk factors.

Materials and Methods：Pyrosequencing was used to determine methylation degrees of 5 probes targeted methylation CpG sites in pre-diagnostic peripheral leukocyte DNA samples (1-6 samples per case, totally 126 samples) that were contributed 0–13 years before diagnosis from 48 HCC incident cases in comparison to 48 HCC-free controls nested within a cohort study. We used generalized estimating equation to examine the association between methylation and HCC stratified according to 5 intervals prior to the diagnosis of HCC. Receiver operating characteristic curve and area under the curve (AUC) statistics were used to evaluate the discriminatory ability of selected biomarkers by time before diagnosis. Linear mixed model was used to establish the association of methylation with HCC risk factors across time.

Results：Methylation levels of selected biomarkers are not only associated with HCC but also stable across 5 time intervals prior to the diagnosis. Furthermore, AUC of selected candidate methylation markers are unchanged by time before diagnosis. On the other hand, according to individual trend, DNA methylation levels in 15 HCC participants are increased by time before diagnosis < 3 years. Finally, HCC risk factors such as HBeAg and cigarette smoking may have an effect on DNA methylation changes across time.

Conclusion：Dynamic change of DNA methylation in selected candidate biomarkers during transition from asymptomatic HBV carrier state to HCC are not consistency among HCC patients so that dynamics of DNA methylation in these HCC patients look like unchangeable. In addition, exposure of putative HCC risk factors may affect dynamic change of DNA methylation.

並列關鍵字

hepatitis B ； hepatocellular carcinoma ； DNA methylation ； dynamic change

參考文獻

1. Torre, L.A., et al., Global cancer statistics, 2012. CA Cancer J Clin, 2015. 65(2): p. 87-108.

2. 衛生福利部國民健康署＿民國101年癌症登記報告.

3. Parkin, D.M., The global health burden of infection-associated cancers in the year 2002. Int J Cancer, 2006. 118(12): p. 3030-44.

4. CJ, C., Y. MW, and L. YF, Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol Hepatol, 1997. 12(9-10): p. S294-308.

5. RP, B., et al., Hepatocellular carcinoma and hepatitis B virus A prospective study of 22,707 men in Taiwan. Lancet, 1981. 2(8256): p. 1129-1133.