[Demethylation in the promoter area by the antisense of human DNA MTase gene].
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 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.


CONCLUSIONS
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.
Zhang
Hong
H
Department of Oncology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Xiao
Wenhua
W
Liang
Houjie
H
Fang
Dianchun
D
Yang
Shiming
S
Luo
Yuanhui
Y
D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D018899:CpG Islands; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D016373:DNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015820:Cadherins; D016373:DNA, Antisense; D012333:RNA, Messenger; D004247:DNA; D015254:DNA Modification Methylases