Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.
Hepatology. 2012 Nov;56(5):1817-27. doi: 10.1002/hep.25870. Epub 2012 Oct 14.
To identify new tumor-suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome-wide methylation profiling and vertical integration with array-based comparative genomic hybridization (aCGH), as well as expression data from a cohort of well-characterized human hepatocellular carcinomas (HCCs). Bisulfite-converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy-number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5-aza-2'-deoxycytidine (5-aza-dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor-suppressor gene (period homolog 3 [PER3], insulin-like growth-factor-binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down-regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5-aza-dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor-suppressive function for PER3 and IGFALS in vitro.
The present study illustrates that vertical integration of methylation data with high-resolution genomic and transcriptomic data facilitates the identification of new tumor-suppressor gene candidates in human HCC.
为了鉴定与人类肝癌发生相关的新抑癌基因候选者，我们进行了全基因组甲基化谱分析，并与基于阵列的比较基因组杂交（aCGH）以及一组经过充分特征描述的人类肝细胞癌（HCC）的表达数据进行垂直整合。从 63 例 HCC 和 10 例健康对照肝脏中提取的 bisulfite 转化 DNA 用于分析超过 14000 个基因的甲基化状态。在确定 HCC 中差异甲基化的基因后，我们整合了 aCGH 数据确定的其 DNA 拷贝数改变，并将其与基因表达相关联，以鉴定可能因启动子超甲基化而沉默的基因。通过焦磷酸测序进一步证实候选基因的异常甲基化，并用 5-氮杂-2'-脱氧胞苷（5-aza-dC）处理来确定沉默的甲基化依赖性。甲基化谱分析显示 2226 个 CpG 位点在健康对照肝脏和 HCC 之间显示出甲基化差异。其中，537 个 CpG 位点在肿瘤 DNA 中呈高甲基化，而 1689 个位点显示启动子低甲基化。高甲基化组富含已知被多梳抑制复合物 2 失活的基因，而低甲基化组富含印迹基因。我们鉴定了三个符合我们对抑癌基因选择标准的基因（PER3、胰岛素样生长因子结合蛋白、酸性不稳定亚基[IGFALS]和蛋白 Z）。与肿瘤周围和健康肝组织相比，PER3 在人类 HCC 中下调。5-aza-dC 处理可恢复 HCC 细胞系中 PER3 的表达，表明启动子超甲基化确实导致基因沉默。此外，功能分析支持 PER3 和 IGFALS 在体外具有肿瘤抑制功能。
本研究表明，将甲基化数据与高分辨率基因组和转录组数据进行垂直整合有助于鉴定人类 HCC 中的新抑癌基因候选者。
