RRC ID 44575
Author Iwaki J, Kikuchi K, Mizuguchi Y, Kawahigashi Y, Yoshida H, Uchida E, Takizawa T.
Title MiR-376c down-regulation accelerates EGF-dependent migration by targeting GRB2 in the HuCCT1 human intrahepatic cholangiocarcinoma cell line.
Journal PLoS One
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.
Volume 8(7)
Pages e69496
Published 2013-1-1
DOI 10.1371/journal.pone.0069496
PII PONE-D-13-09943
PMID 23922722
PMC PMC3724868
MeSH Bile Duct Neoplasms / genetics Bile Duct Neoplasms / pathology Bile Ducts, Intrahepatic Cell Line, Tumor Cell Movement / drug effects Cell Movement / genetics* Cholangiocarcinoma / genetics* Cholangiocarcinoma / pathology* DNA Methylation / drug effects DNA Methylation / genetics Down-Regulation / drug effects Down-Regulation / genetics* Epidermal Growth Factor / pharmacology* Epigenesis, Genetic / drug effects GRB2 Adaptor Protein / metabolism* Gene Expression Regulation, Neoplastic / drug effects Gene Regulatory Networks / drug effects Gene Regulatory Networks / genetics Humans Liver Neoplasms / genetics* Liver Neoplasms / pathology* MicroRNAs / genetics* MicroRNAs / metabolism Neoplasm Proteins / metabolism Proteomics Reproducibility of Results
IF 2.74
Times Cited 41
Human and Animal Cells TKKK(RCB1907) HuH-28(RCB1943) TFK-1(RCB2537)