| 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
|
|
WOS Category
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| Human and Animal Cells |
TKKK(RCB1907)
HuH-28(RCB1943)
TFK-1(RCB2537) |
