MiR-376c down-regulation accelerates EGF-dependent migration by targeting GRB2 in the HuCCT1 human intrahepatic cholangiocarcinoma cell line.
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.
Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan.
Jun
J
Iwaki
Kunio
K
Kikuchi
Yoshiaki
Y
Mizuguchi
Yutaka
Y
Kawahigashi
Hiroshi
H
Yoshida
Eiji
E
Uchida
Toshihiro
T
Takizawa
D001650:Bile Duct Neoplasms / Q000235:genetics / Q000473:pathology; D001653:Bile Ducts, Intrahepatic; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics*; D018281:Cholangiocarcinoma / Q000235:genetics* / Q000473:pathology*; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D044127:Epigenesis, Genetic / Q000187:drug effects; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D053263:Gene Regulatory Networks / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D040901:Proteomics; D015203:Reproducibility of Results
D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C573498:MIRN376C microRNA, human; D035683:MicroRNAs; D009363:Neoplasm Proteins; D004815:Epidermal Growth Factor