MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

Jun Iwaki,Yutaka Kawahigashi,Hiroshi Yoshida,Eiji Uchida,Toshihiro Takizawa,Yoshiaki Mizuguchi,Kunio Kikuchi,Noriko Gotoh

doi:10.1371/journal.pone.0069496

Abstract

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.

Highlights

Deep sequencing and transcriptome analysis revealed the existence of non-coding RNAs in mammalian cells [1,2,3]
We revealed that epigenetic repression of miR-376c accelerated epidermal growth factor (EGF)-dependent cell migration by targeting growth factor receptor-bound protein 2 (GRB2) in HuCCT1 cells
We focused on functional studies of miR-376c, which is significantly downregulated in CC cell lines

Summary

Introduction

Deep sequencing and transcriptome analysis revealed the existence of non-coding RNAs (ncRNAs) in mammalian cells [1,2,3]. MicroRNAs (miRNAs) are single-stranded 19- to 25nucleotide ncRNAs that play a critical role in posttranscriptional gene regulation. Downregulation of miR-15 and miR-16 in most chronic lymphocytic leukemia cells leads to upregulation of anti-apoptotic B cell lymphoma 2 (Bcl-2) protein [8]. The increased levels of Ras protein in lung cancer cells leads to upregulated cell growth. The miR-200 family and miR-205 target zinc finger homeodomain enhancer-binding protein (ZEB) transcription factors, which are known to be inducers of the epithelial-mesenchymal transition in breast cancer [9]. Downregulation of these miRNAs are likely to be an essential early step in breast cancer metastasis

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Results

Conclusion