Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor arising from the biliary tract epithelium. Interleukin-6 (IL-6) is a major mediator of inflammation and contributor to carcinogenesis within the biliary tree. Previous studies suggested that enforced IL-6 contributes to cholangiocarcinogenesis through hypermethylation of several genes implicated in CCA. However, the precise mechanisms of IL-6 effects in CCA remain unclear. We now demonstrate that microRNA (miR)-370 is underexpressed in a large cohort of human CCA vs. normal liver tissues. In addition, we show that IL-6 induces a time-dependent silencing of miR-370. In addition, demethylation of CCA cells results in upregulation of miR-370. Furthermore, we demonstrate that miR-370 is imprinted, and that the Intergenic Differentially Methylated Region (IG-DMR) responsible for imprinting regulation of this genomic locus is hypermethylated in response to IL-6 treatment. In addition, the IG-DMR is hypermethylated in human CCA specimens compared to normal matched controls, in the same location as the IL-6 induced hypermethylation. Finally, miR-370 was found to regulate WNT10B in luciferase as well as western blotting experiments. Our data indicate that the paternal allele of miR-370 is normally silenced through genomic imprinting and that the overexpression of IL-6 in CCA effectively suppresses the expression of miR-370 from the maternal allele, lending support to the theory that miR-370 silencing in human CCA follows a classic two-hit mechanism.

胆管癌（Cholangiocarcinoma, CCA）是一类起源于胆道上皮的高致死性恶性肿瘤。白细胞介素-6（Interleukin-6, IL-6）是炎症反应的核心介导因子，同时也是胆道系统癌变的重要促发因素。既往研究指出，过表达的IL-6可通过使与CCA相关的多个基因发生高甲基化，进而促进胆管癌发生发展，但IL-6在CCA中发挥调控作用的具体分子机制仍未明确。本研究证实，相较于正常肝组织，大样本人类CCA队列中微小RNA（microRNA, miRNA）-370呈低表达状态；同时发现IL-6可诱导miR-370发生时间依赖性的基因沉默。对胆管癌细胞进行去甲基化处理可上调miR-370的表达水平。进一步研究表明，miR-370存在基因组印记现象，负责调控该基因组位点印记的基因间差异甲基化区域（Intergenic Differentially Methylated Region, IG-DMR）在经IL-6处理后发生了高甲基化。此外，相较于匹配的正常对照组织，人类CCA标本中的IG-DMR同样发生高甲基化，且该位点与IL-6诱导的高甲基化位点完全一致。最后，通过荧光素酶报告基因实验及蛋白质印迹（western blotting）实验证实，miR-370可调控WNT10B的表达。本研究数据显示，miR-370的父本等位基因通常通过基因组印记机制发生沉默，而CCA中过表达的IL-6可有效抑制母本等位基因的miR-370表达，这一发现支持了“人类CCA中miR-370的沉默遵循经典二次打击（two-hit）机制”这一理论。