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

Fangmei An,Anne C Ferguson-Smith,Esteban Mezey,Masaharu Ishida,Florin M Selaru,Yuriko Mori,Timothy M Pawlik,Gregory J Gores,Lewis R Roberts,Sarah Allen,Qing Xie,Sumitaka Yamanaka,William B Coleman

doi:10.1371/journal.pone.0045606

Fangmei An, Anne C Ferguson-Smith + Show 11 more

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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.

Highlights

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary tract [1]
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
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

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Introduction

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary tract [1]. CCAs are usually diagnosed late in their progression, and the patient survival is usually measured in months [2]. IL-6 is a recognized mitogen and survival factor in human CCA and can contribute to tumor pathogenesis or progression [3]. It appears that elucidation of pathways downstream of IL-6 merits further investigation. Epigenetic regulation of tumor suppressor genes by DNA hypermethylation has been recognized as an important mechanism in tumorigenesis [6]. Imprinting demonstrated the importance of epigenetic control of gene expression both in normal circumstances (embryologic development) as well as in pathological conditions, such as cancer [9]. Loss of imprinting was first linked to cancer in 1993, when overexpression to 2-fold of Insulin-like growth factor 2 (Igf2) was identified in Wilms’ tumor [15,16]. Several studies implicated dysregulated imprinting in hepatocellular carcinoma, there are no studies to date to link imprinting to CCA [20,21]

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