Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker
Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker
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DOI：
10.1002/ijc.28068
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发表时间：
2013-08-01
影响因子：
6.4
通讯作者：
Lehmann, Ulrich
中科院分区：
文献类型：
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作者：
Anwar, Sumadi Lukman;Albat, Cord;Lehmann, Ulrich
据报道，在各种人类恶性肿瘤中，许多microRNA基因因DNA甲基化异常而发生表观遗传失活。然而，对肝细胞癌（HCC）中microRNA基因甲基化的了解相对较少。因此，开始了对HCC中异常高甲基化microRNA基因的系统筛选。分析了肝癌细胞系（n=7）、永生化肝细胞（n=2）和正常肝脏样本（n=5）中39个基因间CpG岛相关microRNA基因的甲基化状态。随后，采用焦磷酸测序技术分析了原发性人HCC样本（n=40）、良性肝肿瘤样本（n=15）和邻近肝组织中13个差异甲基化的microRNA基因。采用实时定量聚合酶链反应（RT-PCR）检测microRNA基因的表达。此外，在DNMT1敲除或DNMT抑制后，检测DNA甲基化和microRNA基因的表达。异常高甲基化和伴随的基因间microRNA基因表达降低是人类HCC中的常见事件：hsa-mir-9-2（23%）、hsa-mir-9-3（50%）、hsa-mir-124-1（20%）、hsa-mir-124-2（13%）、hsa-mir-124-3（43%）、hsa-mir-129-2（58%）、hsa-mir-596（28%）和hsa-mir-1247（38%）。总的来说，它影响了研究中90%的HCC标本。在肝细胞腺瘤（n=10）和局灶性结节增生（n=5）中未发现MicroRNA基因甲基化。DNMT1敲低或DNMT抑制可降低microRNA基因甲基化并刺激表达。在原发性人HCC标本中，高甲基化与microRNA基因的表达呈负相关。三个或更多microRNA基因的一致性高甲基化是HCC检测和预后不良的高度特异性标志物。
Epigenetic inactivation by aberrant DNA methylation has been reported for many microRNA genes in various human malignancies. However, relatively little is known about microRNA gene methylation in hepatocellular carcinoma (HCC). Therefore, a systematic screen for identification of aberrantly hypermethylated microRNA genes in HCC was initiated. The methylation status of 39 intergenic CpG island associated microRNA genes was analyzed in HCC cell lines (n=7), immortalized hepatocytes (n=2) and normal liver samples (n=5). Subsequently, 13 differentially methylated microRNA genes were analyzed in primary human HCC samples (n=40), benign liver tumors (n=15) and the adjacent liver tissues employing pyrosequencing. Expression of microRNA genes was measured using quantitative real-time polymerase chain reaction (RT-PCR). In addition, DNA methylation and expression of microRNA genes were measured after DNMT1 knockdown or DNMT inhibition. Aberrant hypermethylation and concomitant reduction in expression of intergenic microRNA genes is a frequent event in human HCC: hsa-mir-9-2 (23%), hsa-mir-9-3 (50 %), hsa-mir-124-1 (20%), hsa-mir-124-2 (13%), hsa-mir-124-3 (43%), hsa-mir-129-2 (58%), hsa-mir-596 (28%) and hsa-mir-1247 (38%). Altogether, it affects 90% of the HCC specimens under study. MicroRNA gene methylation is not found in hepatocellular adenoma (n=10) and focal nodular hyperplasia (n=5). DNMT1 knockdown or DNMT inhibition reduced microRNA gene methylation and stimulated expression. In primary human HCC specimens hypermethylation and expression of microRNA genes showed an inverse correlation. Concordant hypermethylation of three or more microRNA genes is a highly specific marker for the detection of HCC and for poor prognosis.