title: si-DNMT1 restore tumor suppressor genes expression through the reversal of DNA hypermethylation in cholangiocarcinoma.
abstract: OBJECTIVE
  The aim of our study was to evaluate the effect of shorthairpin RNA plasmid vector knockdown of human DNA methyltransferase 1 on proliferation and the methylation status and expression of tumor suppressor genes in hilar cholangiocarcinoma.
  
  
  METHODS
  The hilar cholangiocarcinoma cell line QBC939 was utilized for this study. QBC939 cells were transfected with a shorthairpin RNA plasmid vector targeting human DNA methyltransferase 1. Control and human DNA methyltransferase 1 shorthairpin RNA plasmid vector-transfected cells were collected at different time points, and the expression levels of human DNA methyltransferase 1 and tumor suppressor genes (cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1) were detected by reverse transcription-polymerase chain reaction. Furthermore, interfering efficiency was confirmed by Western blotting. The methylation status of tumor suppressor genes was detected using methylation-specific polymerase chain reaction. Furthermore, the effect of human DNA methyltransferase 1 knockdown on proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
  
  
  RESULTS
  Targeted gene knockout of human DNA methyltransferase 1 restored the expression levels of tumor suppressor genes cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1, indicating that the silencing of these tumor suppressor genes is associated with promoter hypermethylation. In addition, knockdown of human DNA methyltransferase 1 expression significantly inhibited the proliferation of QBC939 cells.
  
  
  CONCLUSIONS
  Targeted knockdown of human DNA methyltransferase 1 expression restores the expression levels of tumor suppressor genes, thus inhibiting the proliferation of QBC939 cells. These results may provide insight for the development of novel therapies for cholangiocarcinoma.
authors.affiliation: Department of Hepatobiliary Sugery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Xiang
authors.affiliation: Department of Hepatobiliary Sugery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. Electronic address: luofangdoctor19@163.com.
authors.forename: Fang
authors.initials: F
authors.lastname: Luo
authors.affiliation: Department of Hepatobiliary Sugery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Hepatobiliary Sugery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Fangyu
authors.initials: F
authors.lastname: Zhu
authors.affiliation: Department of Hepatobiliary Sugery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jiming
authors.initials: J
authors.lastname: Wang
mesh_terms: D001650:Bile Duct Neoplasms / Q000235:genetics*; D015820:Cadherins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018281:Cholangiocarcinoma / Q000235:genetics*; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D019175:DNA Methylation; D055786:Gene Knockout Techniques; D006801:Humans; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D015820:Cadherins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; C411744:DMAP1 protein, human; C411762:RASSF1 protein, human; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins