title: Genetic and epigenetic alterations are involved in the regulation of             TPM1 in cholangiocarcinoma.
abstract: Cholangiocarcinoma is a malignant tumor originating from biliary epithelial             cells. The tumor suppressor gene tropomyosin 1 (TPM1) is downregulated in several             human cancer types; however, its expression status in cholangiocarcinoma is still             unknown. We elucidated TPM1 expression and its regulation mechanism in cholangiocarcinoma.             Real-time (RT)-PCR, western blot analysis and immunohistochemistry were performed             to examine TPM1 expression levels in cholangiocarcinoma cell lines and tumor tissues.             Cell lines were treated with lentiviral vector containing the miR-21 knockdown             and inhibitors of genetic and epigenetic mechanisms (manumycin A, LY294002, U0126,             DAC and TSA), and the TPM1 expression change was observed by RT-PCR and western             blot analyses. Cell proliferation, apoptosis and migration were evaluated by water-soluble             tetrazolium salt (WST-1) assay, flow cytometry and wound healing experiments,             respectively. TPM1 was downregulated in the intrahepatic cholangiocarcinoma cells             (HuCCT1) and upregulated in the extrahepatic cholangiocarcinoma cells (QBC939)             compared with normal intrahepatic biliary epithelial cells (HIBEC). TPM1 stained             negative in the intrahepatic cholangiocarcinoma tissues, as revealed by immunohistochemistry,             although there was no significant difference in staining of the intrahepatic cholangiocarcinoma             tissues and adjacent non-cancer tissues. RAS and two important downstream signaling             pathways (RAS/PI3K/AKT and RAS/MEK/ERK) were involved in TPM1 regulation and inhibition             of the epigenetic mechanisms such as DNA methylation, histone deacetylation and             miR-21 upregulation upregulated TPM1 expression. Inhibitors of genetic and epigenetic             mechanisms (manumycin A, LY294002, U0126, DAC and TSA) inhibited cell proliferation             and migration and induced apoptosis. These data indicated that TPM1 is downregulated             in HuCCT1 cells and that the Ras signaling pathway as well as DNA methylation,             histone deacetylation and miR-21 upregulation play important roles in the suppression             of TPM1 expression in HuCCT1 cells. Thus, compounds that inhibit genetic and epigenetic             mechanisms may be promising agents in treating cholangiocarcinoma.
authors.affiliation: Department of Minimally Invasive Surgery, The First Affiliated                     Hospital of Nanjing Medical University, Nanjing 210029, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: Kedong
authors.initials: K
authors.lastname: Li
authors.forename: Haofeng
authors.initials: H
authors.lastname: Liu
authors.forename: Yueming
authors.initials: Y
authors.lastname: Sun
mesh_terms: D017209:Apoptosis; D001650:Bile Duct Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018281:Cholangiocarcinoma / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; C490775:TPM1 protein, human; D014335:Tropomyosin; D019869:Phosphatidylinositol 3-Kinases