找尋B型肝炎帶原者發展成肝細胞癌相關之病毒與宿主免疫交互作用的表遺傳標記

Abstract

背景與目的 B型肝炎帶原者發展成肝細胞癌的原因相當複雜，目前對病毒因子作用的了解已有迅速發展，但病毒因子與宿主免疫反應之間的交互作用仍然未知。因病毒感染及發炎反應均會影響甲基化的程度，因此DNA甲基化標記可反映病毒與宿主之交互作用，並具有調節功能改變和臨床結果的效能。我們使用晶片及焦磷酸定序來評估與病毒因子和肝細胞癌相關的甲基化位點，並試圖確定與B型肝炎帶原者中肝細胞癌進程相關的病毒與宿主之交互作用。 材料與方法 從4,841個B型肝炎表面抗原陽性男性的世代研究獲得周邊血液白血球的DNA。 首先，我們在巢式病例對照研究(n = 96)中使用Infinium HumanMethylation450晶片進行了表遺傳全基因組的分析，檢驗甲基化程度與血液中B型肝炎病毒DNA及肝細胞癌的關聯。其次，我們進行了基因功能與富集分析並鑑定表遺傳變化與基因表達的關聯，藉以確認功能的相關性。第三，在較大的巢式病例對照研究中，利用焦磷酸定序驗證選出之甲基化位點(n = 476)。最後，使用DNA甲基化標記進行中介分析，以檢驗B型肝炎帶原者肝細胞癌發展過程中病毒與宿主交相作用的潛在重要性。 結果 通過表遺傳全基因組分析，挑選出在B型肝炎病毒相關免疫途徑中四個不同基因上的五支探針，其中四支探針特定位點的甲基化程度與基因表達強度的變化顯著相關。利用較大的巢式病例對照研究驗證了三支探針(位於IFI44L基因的cg05696877及cg06872964和位於PRF1基因的cg22900360)與病毒量存在顯著相關。中介分析顯示，病毒量解釋了IFI44L和PRF1甲基化對肝細胞癌的部分影響，且IFI44L基因的甲基化也是病毒量與肝細胞癌、B型肝炎病毒基因型與病毒量/e抗原之間關係的中介，結果還顯示PRR5L甲基化會因透過對IFNG/PRF1甲基化和病毒量的影響而與肝細胞癌相關。 結論 我們的結果表明，重要的B型肝炎病毒相關免疫途徑中的免疫基因甲基化可能是慢性B型肝炎病毒感染和肝細胞癌之間的起始或中介。然而，未來需要進行更複雜的功能研究以闡明潛在的機制。

Background and Aims In hepatitis B virus (HBV)-related hepatocellular carcinoma(HCC), understanding of viral factors have been progressed rapidly, but the interaction between viral factors and host immune response remains largely unknown. Both viral infection and inflammation can affect the extent of methylation, and thus DNA methylation markers could reflect the virus-host interaction, which can mediate functional changes and clinical outcomes. We used microarray and pyrosequencing to assess methylation sites related to viral factors and HCC, and attempted to determine the virus-host interaction associated with the development of HCC in HBV carriers. Materials and Methods Peripheral leukocyte DNA were obtained from a longitudinal cohort of 4,841 hepatitis B surface antigen-positive men. First, we conducted an epigenome-wide analysis with the Infinium HumanMethylation450 array in a nested case-control study(n=96). Methylation levels were examined for associations with circulating HBV DNA and HCC. Second, we conducted gene sets enrichment analysis and correlated identified epigenetic changes with gene expression to investigate functional relevance of the findings. Third, candidate methylation sites were taken forward for replication with pyrosequencing in a larger nested case-control study (n=476). Finally, statistical mediation analysis with DNA methylation markers was used to examine potential importance of virus-host interaction during the development of HBV-related HCC. Results Through epigenome-wide analysis, five probes across 4 different genes in HBV-related immune pathways. Methylation levels in four of these five probes targeted sites were significantly associated with concurrent changes in gene expression levels. The associations with viral load were confirmed for three probes (cg05696877 and cg06872964 at IFI44L gene and cg22900360 at PRF1 gene) in the replication study. Mediation analysis showed that viral load explained part of the effect of IFI44L and PRF1 methylation on HCC, and methylation in the IFI44L gene was also a mediator of the relation between both viral load and HCC, genotype and viral load/ HBeAg. Our results also showed that the PRR5L methylation was associated with HCC through its effect on IFNG/ PRF1 methylation and viral load. Conclusion Our results suggest that methylation of immune genes in important HBV-related immune pathways may be an initiator or an intermediary between chronic HBV infection and HCC. However, more complex functional studies are warranted in the future to elucidate the underlying mechanisms.