Effect of Transfection with Antisense DNMT3b Gene Eukaryotic Expression Vector on Expression of DNMT3b Gene in Human Cholangiocarcinoma Cell Line_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

ZUO Shi , GUO Wei , LIU Minfeng , DONG Jingqing , XU Lining , LUO Jian , ZOU Shengquan.

Department of General Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

Corresponding author：

Keywords：

DNA methyltransferase 3b Antisense RNA Transfection Gene expression Cholangiocarcinoma

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【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939.
MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively.
ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01).
ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.

Citation： ZUO Shi,GUO Wei,LIU Minfeng,DONG Jingqing,XU Lining,LUO Jian,ZOU Shengquan.. Effect of Transfection with Antisense DNMT3b Gene Eukaryotic Expression Vector on Expression of DNMT3b Gene in Human Cholangiocarcinoma Cell Line. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2006, 13(1): 56-60. doi: Copy

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2. Egger G, Liang G, Aparicio A, et al. Epigenetics in human disease and prospects for epigenetic therapy [Ｊ]. Nature， 2004; 429(6990)∶457.
3. Hermann A, Gowher H, Jeltsch A. Biochemistry and biology of mammalian DNA methyltransferases [Ｊ]. Cell Mol Life Sci， 2004; 61(19-20)∶2571.
4. Sambrook J, Russell DW，黄培堂，等译. 分子克隆实验指南 [M]. 第3版. 北京：科学出版社, 2002∶629～630.
5. Jin F, Dowdy SC, Xiong Y, et al. Upregulation of DNA methyltransferase 3B expression in endometrial cancers [Ｊ]. Gynecol Oncol， 2005; 96(2)∶531.
6. Girault I, Tozlu S, Lidereau R, et al. Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas [Ｊ]. Clin Cancer Res， 2003; 9(12)∶4415.
7. Nagai M, Nakamura A, Makino R, et al. Expression of DNA (5cytosin)methyltransferases (DNMTs) in hepatocellular carcinomas [Ｊ]. Hepatol Res， 2003; 26(3)∶186.
8. Beaulieu N, Morin S, Chute IC, et al. An essential role for DNA methyltransferase DNMT3B in cancer cell survival [Ｊ]. J Biol Chem， 2002; 277(31)∶28176.
9. 唐启彬，孙华文，邹声泉. 5氮2脱氧胞苷体内外抑制胆管癌细胞生长的研究 [Ｊ]. 中华普通外科杂志， 2004； 19（5）∶295.
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11. Beaulieu N, Morin S, Chute IC, et al. An essential role for DNA methyltransferase DNMT3B in cancer cell survival [Ｊ]. J Biol Chem， 2002; 277(31)∶28176.
12. Lee LK, Roth CM. Antisense technology in molecular and cellular bioengineering [Ｊ]. Curr Opin Biotechnol， 2003; 14(5)∶505.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Effect of Transfection with Antisense DNMT3b Gene Eukaryotic Expression Vector on Expression of DNMT3b Gene in Human Cholangiocarcinoma Cell Line

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