Regulation of demethylation and re-expression of RASSF1A gene in gastric cancer cell lines by combined treatment of 5-Aza-CdR and NaB.

AIM

To investigate the changes of methylation state and expression of RASSF1A gene in human gastric cancer cell lines SGC7901 and BGC823 which were treated in vitro with demethlylating agent 5-Aza-CdR in combination with histone deacetylase inhibitor NaB.

METHODS

After SGC7901 and BGC823 cells were treated with 5-Aza-CdR and/or NaB, the methylation state of RASSF1A gene was detected by methylation-specific PCR, and the changes in expression of mRNA and protein level of RASSF1A gene were observed by RT-PCR and Western-blotting before and after drug treatment.

RESULTS

Hypermethylation was detected in the promoter region of RASSF1A gene in both SGC7901 and BGC823 cells, and there was no expression of this gene at both mRNA and protein level. After treatment with 5-Aza-CdR, demethylation occurred in the promoter region of RASSF1A gene, which subsequently induced re-expression of this gene. The treatment with NaB alone showed no effect on the methylation state and expression of RASSF1A gene. The combined treatment of 5-Aza-CdR and NaB induced complete demethylation of RASSF1A gene, leading to a significantly higher re-expression of the mRNA and protein of RASSF1A than those treated with 5-Aza-CdR alone (P<0.05).

CONCLUSION

Hypermethylation in the promoter region is related to inactivation of RASSF1A gene in human gastric cancer cell lines SGC7901 and BGC823, while demethlylating agent 5-Aza-CdR can reverse the methylation state of RASSF1A gene and induce its re-expression. Histone deacetylase inhibitor NaB had a synergistic effect with 5-Aza-CdR in both demethylation and gene transcriptional regulation.