Demethylation in the promoter area by the antisense of human DNA MTase gene
Zhang, H.; Xiao, W.; Liang, H.; Fang, D.; Yang, S.; Luo, Y.
Zhonghua Zhong Liu Za Zhi 24(5): 444-447
2002
ISSN/ISBN: 0253-3766 PMID: 12485494 Document Number: 549023
Objective: To investigate the change in the expression of E-Cadherin of human hepatocarcinoma cell line SMMC-7721 after transfection by antisense human DNA MTase gene. Methods: DNA MTase gene eukaryotic expression vectors, including sense and antisense fragments, were constructed with recombinant technology and transfected into the hepatocarcinoma cell line SMMC-7721 with liposome DOTAP. The expression of DNA MTase gene mRNA and E-Cadherin gene mRNA was examined with RT-PCR and the expression of E-Cadherin with immunohistochemical method and flow cytometry. The status of methylation in E-Cadherin gene promoter area was examined with methylation specific PCR (MSP). Results: The sense and antisense eukaryotic expression vectors were successfully constructed and then the constructed recombinant plasmids were successfully transfected into SMMC-7721 cell with liposome DOTAP. The expression of endogenous DNA MTase mRNA was obviously decreased with E-Cadherin gene mRNA and its activity increased in the SMMC-7721 cell, which was tranfected with antisense DNA MTase gene fragment. Moreover, demethylation in the promoter area of E-Cadherin gene was observed with MSP. Conclusion: Demethylation in the promoter area and increasing mRNA level of E-Cadherin gene can be induced by expression inhibition of DNA MTase gene of SMMC-7721 cell line.