Department of Biliary-Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology 1095, Jiefang Road, Wuhan 430030, PR China.
Oncol Rep. 2011 Feb;25(2):443-51. doi: 10.3892/or.2010.1089. Epub 2010 Dec 9.
Aberrant expression of miRNAs is associated with particular cancers showing tissue- and clinical-feature-specificity patterns. Some miRNA genes harboring or being embedded in CpG islands undergo methylation mediated silencing. MBP, methyl CpG binding protein, suppresses transcription through binding to methylated CpG dinucleotides. Expression of miR-373 has been reported to be suppressed in malignant bile duct cell lines. Bioinformatic prediction reveals that the transcription start site (TSS) of miR-373 is implanted in a 402 bp canonical CpG island containing 26 CpG dinucleotides. In this study, we aim to determine the epigenetic regulation of miR-373 gene in hilar cholangiocarcinoma. Taqman microRNA assay shows that down-regulation of miR-373 is closely associated with poor cell differentiation, advanced clinical stage and shorter overall and disease-free survival in hilar cholangiocarcinomas. Methylation analysis shows that the promoter-associated CpG island is hypermethylated which is consistent with the inhibition of miR-373. Chromatin immunoprecipitation (ChIP) assay indicates that down-regulation of miR-373 results from the selective recruitment of MBD2 to methylated CpG islands. In contrast, MBD2 knock-down by use of a specific siRNA promoted the expression of miR-373. Reactivation of miR-373 by pharmacologic induction of 5-aza-CdR and trichostatin A (TSA) led to decreased enrichment of MBD2 at CpG island regions. Enhanced expression of exogenous MBD2 in stable QBC939 cells which stably express pGL4-m373-prom induces strengthened recruitment of MBD2. Our findings suggest that miR-373 is a methylation-mediated gene and the implication of MBD2 in methylation-mediated suppression of miR-373 plays an important role in tumourigenesis and development in hilar cholangiocarcinoma.
miRNA 的异常表达与具有组织和临床特征特异性模式的特定癌症有关。一些含有 CpG 岛或嵌入 CpG 岛的 miRNA 基因通过甲基化介导的沉默而失活。甲基 CpG 结合蛋白（MBP）通过与甲基化的 CpG 二核苷酸结合来抑制转录。已经报道 miR-373 的表达在恶性胆管细胞系中受到抑制。生物信息学预测表明，miR-373 的转录起始位点（TSS）位于一个含有 26 个 CpG 二核苷酸的 402 bp 经典 CpG 岛中。在本研究中，我们旨在确定 miR-373 基因在肝门部胆管癌中的表观遗传调控。Taqman 微 RNA 检测显示，miR-373 的下调与细胞分化不良、临床分期较晚以及肝门部胆管癌患者总生存期和无病生存期较短密切相关。甲基化分析显示，启动子相关的 CpG 岛呈高甲基化状态，这与 miR-373 的抑制一致。染色质免疫沉淀（ChIP）检测表明，miR-373 的下调是由于 MBD2 选择性募集到甲基化的 CpG 岛。相比之下，使用特异性 siRNA 敲低 MBD2 可促进 miR-373 的表达。通过 5-氮杂胞苷和曲古抑菌素 A（TSA）的药物诱导使 miR-373 重新激活，导致 MBD2 在 CpG 岛区域的富集减少。在稳定表达 pGL4-m373-prom 的 QBC939 细胞中过表达外源 MBD2 可诱导 MBD2 更强的募集。我们的研究结果表明，miR-373 是一种受甲基化调控的基因，MBD2 在甲基化调控 miR-373 抑制中的作用在肝门部胆管癌的发生和发展中起着重要作用。
