Effect of antisense DNMT3b gene eukaryotic expression plasmid on expression of the DNMT3b gene in human biliary tract carcinoma cells.
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 neoR 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 post-transcription levels in the expression of the DNMT3b gene between the non-transfection group and the antisense DNMT3b gene eukaryotic expression plasmid transfection group (P<0.01).


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 carcinoma.
Department of General Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
Shi
S
Zuo
Jing-Qing
JQ
Dong
Wei
W
Guo
Min-Feng
MF
Liu
Li-Ning
LN
Xu
Jian
J
Luo
Sheng-Quan
SQ
Zou
D001661:Biliary Tract Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D019175:DNA Methylation; D005057:Eukaryotic Cells / Q000378:metabolism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D066298:In Vitro Techniques; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics*; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000097584:DNA Methyltransferase 3B
D004248:DNA (Cytosine-5-)-Methyltransferases; D016372:RNA, Antisense; D012334:RNA, Neoplasm; D000097584:DNA Methyltransferase 3B