Unique Association between Global DNA Hypomethylation and Chromosomal Alterations in Human Hepatocellular Carcinoma

Global DNA hypomethylation is a characteristic feature of cancer cells that closely associates with chromosomal instability (CIN). However, the association between these characteristics during hepatocarcinogenesis remains unclear. Herein, we determined the relationship between hypomethylation and CIN in human hepatocellular carcinoma (HCC) by analyzing 179 HCCs, 178 matched non-tumor livers and 23 normal liver tissues. Hypomethylation at three different repetitive DNA (rDNA) sequences and hypermethylation of 12 CpG loci, including 11 tumor suppressor gene (TSG) promoters, were quantified using MethyLight or combined bisulfite restriction analysis. Fractional allelic loss (FAL) was used as a marker for CIN, calculated by analyzing 400 microsatellite markers. Gains and losses at each chromosome were also determined using semi-quantitative microsatellite analysis. The associations between rDNA hypomethylation and FAL, as well as between TSG hypermethylation and FAL were investigated. Significantly more hypomethylation was observed in HCC tissues than in normal liver samples. Progression of hypomethylation during carcinogenesis was more prominent in hepatitis C virus (HCV)-negative cases, which was in contrast to our previous reports of significantly increased TSG methylation levels in HCV-positive tumors. Absence of liver cirrhosis and higher FAL scores were identified as independent contributors to significant hypomethylation of rDNA in HCC. Among the chromosomal alterations frequently observed in HCC, loss of 8p, which was unique in the earliest stages of hepatocarcinogenesis, was significantly associated with hypomethylation of rDNA by multivariable analysis (p = 0.0153). rDNA hypomethylation was also associated with a high FAL score regardless of tumor differentiation (p = 0.0011, well-differentiated; p = 0.0089, moderately/poorly-differentiated HCCs). We conclude that DNA hypomethylation is an important cause of CIN in the earliest step of HCC, especially in a background of non-cirrhotic liver.

全基因组DNA低甲基化是癌细胞的标志性特征之一，其与染色体不稳定性（chromosomal instability, CIN）密切相关。然而，二者在肝癌发生过程中的关联机制仍未明确。本研究通过分析179例肝细胞癌（hepatocellular carcinoma, HCC）样本、178例配对癌旁肝组织及23例正常肝组织，明确了HCC中低甲基化与CIN的关联。本研究采用MethyLight技术或联合亚硫酸氢盐限制性内切酶分析，对三类不同的重复DNA（rDNA）序列的低甲基化水平，以及包含11个肿瘤抑制基因（tumor suppressor gene, TSG）启动子在内的12个CpG位点的高甲基化水平进行了定量检测。本研究以等位基因缺失分数（fractional allelic loss, FAL）作为CIN的检测指标，通过分析400个微卫星标记计算得到FAL值；同时采用半定量微卫星分析技术检测各染色体的拷贝数增减情况。本研究同时分析了rDNA低甲基化与FAL、TSG高甲基化与FAL之间的关联。研究发现，肝癌组织中的低甲基化水平显著高于正常肝组织样本。在未感染丙型肝炎病毒（hepatitis C virus, HCV）的病例中，肝癌发生过程中的低甲基化进展更为显著，这与我们此前报道的“HCV阳性肿瘤中TSG甲基化水平显著升高”的结论恰好相反。无肝硬化背景及较高的FAL评分被确定为HCC中rDNA显著低甲基化的独立影响因素。在HCC常见的染色体改变中，8p染色体缺失（这一改变仅见于肝癌发生的最早期阶段）经多变量分析证实与rDNA低甲基化显著相关（p=0.0153）。无论肿瘤分化程度如何，rDNA低甲基化均与高FAL评分显著相关（高分化HCC：p=0.0011；中/低分化HCC：p=0.0089）。综上，本研究表明DNA低甲基化是HCC发生最早期阶段CIN的重要诱因，尤其在无肝硬化的肝组织背景下更为显著。