Effect of antisense DNMT3b gene eukaryotic expression plasmid on expression of the DNMT3b gene in human biliary tract carcinoma cells

作     者：Shi Zuo, Jing-Qing Dong, Wei Guo, Min-Feng Liu, Li-Ning Xu, Jian Luo and Sheng-Quan Zou Department of General Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China 

作者机构：Department of General Surgery Affiliated Tongji Hospital Tongji Medical College Huazhong University of Science & Technology Wuhan 430030 China. 

出 版 物：《Hepatobiliary & Pancreatic Diseases International》 (国际肝胆胰疾病杂志（英文版）)

年 卷 期：2006年第5卷第1期

页      面：123-128页

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

基　　金：This study was supported by a grant from Hi-Tech Research and Development Program of China (863 Program) (No. 2002AA214061) 

主　　题：DNA methyltransferase 3b antisense RNA transfection gene expression biliary tract carcinoma 

摘      要：BACKGROUND: Hypermethylation of the promoter region is one of the major mechanisms of tumor suppressor gene inactivation. DNA methyltransferase 3b (DNMT3b), an enzyme that participates in the establishment of de novo methylation patterns, is highly expressed in many tumor cells and tissues, and it is closely associated with hypermethylation of the promoter of tumor suppressor genes. The aim of this study was to explore the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the expression of the DNMT3b gene in human biliary tract carcinoma cell. METHODS: The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into the human biliary tract carcinoma cell line QBC-939 with lipofectamine transfection reagent, and positive cell clones were formed using G418 selection after transfection. The constructed recombinant plasmid was transfected into QBC-939 cells successfully and was confirmed by amplification of the exogenous neo^R gene with the polymerase chain reaction method. The expression of DNMT3b gene mRNA and protein was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry respectively. RESULTS: Following transfection, the mRNA level of the DNMT3b gene decreased from 0.956±0.053 to 0.209±0.023, and the protein level of the DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. Very significant differences were observed both at the transcription and posttranscription levels in the expression of the DNMT3b gene between the non-tranfection group and the antisense DN- MT3b gene eukaryotic expression plasmid transfection group (P0.01). CONCLUSIONS: Transfection with the antisense DNMT3b gene eukaryotic expression plasmid can significantly reduce the expression level of the DNMT3b gene in the human biliary tract carcinoma cell line QBC-939. This study may provide a valid method to investigate the function of the DNMT3b gene and its role in biliary tract c