The Experimental Study Of Reversing The Malignant Phenotype Of The Hepatocellular Carcinoma Cell Line With Antisense DNA MTase Gene

Aims At present study,we transfect the constructed DNA MTase antisense fragment expressing vectors into the hepatocelluer carcinoma cell line SMMC-7721.With the blank control of SMMC-7721 cell line and the negative control of the SMMC-7721 cell line transfected with the constructed DNA MTase sense fragment expressing vector,we observed the alteration of the expression of endogenous DNA MTase gene mRNA,the alteration of methylation status of E-Cadherin gene in promoter region,E-Cadherin gene mRNA and its protein,and the changing of biological behaviors of SMMC-7721 in order to investigate the correlation among aberrant DNA methylation, tumor suppressor gene and hepatocellur carcinoma and the mechanism of hepatocellur carcinoma, and try to find out a new therapeutic method to hepatocelluer carcinoma.Methods The sense and antisense fragments were obtained from RT-PCR by the use of the primers added with restriction site EcoRＩ/XhoＩ,EcoRＩ/XbaＩto the 5ˊrigion.The fragments were inserted into the plasmid vectors pCl-neo in cis or trans direction after digested with the restriction endonuclease using the recombinant technology.The constructed recombinants were identified with restriction endonuclease analyzing and sequencing.The recombinants were transfected into the hepatocelluar carcinoma cell line SMMC-7721 with DOTAP liposomal transfection.The cell lines transfected with pCl-S,pCl-AS were respectively named as7721-S,7721-AS,and the cell line untransfected was named 7721.The alteration of the expression of endogenous DNA MTase mRNA and E-Cadherin gene mRNA were detected with RT-PCR.The methylation status of E-Cadherin gene in the promoter region with methylation-specific PCR(MSP).The cell morphology by phase contrast microscope and transmission electron microscope and confocal laser scanning microscope,growth speed by MTT assay, cell cycle distribution and apoptotic rate and E-Cadherin protein expression by flow cytometry analyses and immunohistochemistry, clones efficiency were examined.Results The constructed sense and antisense DNA MTase gene eukaryotic expression vectors were proved to be the same as designed by restriction endonuclease analysing and sequencing.The recombinants were transfected into SMMC-7721 cell line and expressing the sense and antisense DNA MTase gene fragments.The decreased expression of endogenous DNA MTase mRNA,demethylation of E-Cadherin gene in the promoter region, increased expression of the E-Cadherin mRNA,cutdown atypia,definite inhibition and reversion to the malignancy phenotype of 7721-AS were found.Conclusions Antisense DNA MTase gene can reduce the expression of endogenous DNA MTase gene mRNA of 7721-AS. Antisense DNA MTase gene can induce demethylation of E-Cadherin gene in the promoter region.Antisense DNA MTase gene can increase the expression of tumor suppressor gene E-Cadherin gene mRNA and its protein of 7721-AS. Antisense DNA MTase gene can minish the atypia and make the malignancy phenotype to be inhibited and reversed definitely.The therapeusis of antisense DNA MTase gene has potential value in clinic.