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Effect of antisense DNMT3b gene eukaryotic expression plasmid on expression of the DNMT3b gene in human biliary tract
Shi Zuo1, Jing-Qing Dong, Wei Guo
1Department of General Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
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.
Insights
Antisense DNA methyltransferase 3b (DNMT3b) gene transfection significantly reduced DNMT3b expression in human biliary tract carcinoma cells. This approach offers a method to study DNMT3b
Area of Science:
- Oncology
- Molecular Biology
- Gene Expression Regulation
Background:
- Hypermethylation of tumor suppressor genes is a key mechanism in cancer development.
- DNA methyltransferase 3b (DNMT3b) is highly expressed in tumors and linked to gene silencing.
- Investigating DNMT3b's role in biliary tract carcinoma is crucial.
Purpose of the Study:
- To assess the impact of antisense DNMT3b gene transfection on DNMT3b expression in human biliary tract carcinoma cells.
- To evaluate the efficacy of a specific gene therapy approach targeting DNMT3b.
Main Methods:
- Human biliary tract carcinoma cell line QBC-939 was transfected with an antisense DNMT3b gene eukaryotic expression plasmid.
- Lipofectamine and G418 selection were used for successful transfection and clone formation.
- DNMT3b mRNA and protein levels were quantified using RT-PCR and flow cytometry.
Main Results:
- Antisense DNMT3b transfection markedly decreased DNMT3b mRNA levels from 0.956 to 0.209.
- DNMT3b protein expression significantly reduced from 75.38% to 29.87% post-transfection.
- Statistically significant differences (P<0.01) were observed at both mRNA and protein levels.
Conclusions:
- Antisense DNMT3b gene transfection effectively suppresses DNMT3b gene expression in QBC-939 cells.
- This method provides a valuable tool for exploring DNMT3b's function in biliary tract carcinoma.
- The findings support DNMT3b as a potential therapeutic target in this cancer type.
