微小 RNA-373 与甲基化 CpG 结合域蛋白 2 在肝内胆管癌中的相互调控。
Mutual regulation between microRNA-373 and methyl-CpG-binding domain protein 2 in hilar cholangiocarcinoma.
机构信息
Department of Biliary-pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
出版信息
World J Gastroenterol. 2012 Aug 7;18(29):3849-61. doi: 10.3748/wjg.v18.i29.3849.
AIM
To investigate the reciprocal modulation between microRNA (miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpG-binding domain protein (MBD)2.
METHODS
MiR-373 expression was examined using the TaqMan miRNA assay. Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction, and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay. Mutation analysis was conducted using the QuikChange™ Site-Directed Mutagenesis kit. The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region (3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.
RESULTS
In hilar cholangiocarcinoma, miR-373 decreased and was closely associated with poor cell differentiation, advanced clinical stage, and shorter survival. The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373. MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373. Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island, and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.
CONCLUSION
MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.
目的
通过研究 miR-373 与甲基化 CpG 结合域蛋白 2（MBD2）之间的相关性，探讨 miRNA 与 DNA 甲基化之间的相互调节作用。
方法
采用 TaqMan miRNA 检测法检测 miR-373 的表达情况。采用甲基化特异性聚合酶链反应（PCR）检测 miR-373 的甲基化情况，采用染色质免疫沉淀（ChIP）assay 研究甲基结合蛋白的募集情况。采用 QuikChange™ 定点突变试剂盒进行突变分析。通过双荧光素酶报告基因检测评估 miR-373 基因启动子构建物的活性以及 miR-373 对 MBD2 3'非翻译区（3'UTR）的靶向作用。
结果
在肝内胆管癌中，miR-373 表达下调，与细胞分化不良、临床分期较晚和生存期较短密切相关。miR-373 基因启动子相关 CpG 岛发生超甲基化，抑制 miR-373 的表达。MBD2 上调并富集于 miR-373 基因启动子相关 CpG 岛。MBD2 可促进 miR-373 启动子相关 CpG 岛的甲基化抑制 miR-373 的表达，而 miR-373 则通过靶向 3'UTR 负调控 MBD2 的表达。
结论
miR-373 通过 CpG 岛甲基化的反馈环作为 MBD2 活性的直接转录靶标和负调控因子。
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