In vitro introduction of recombinant trichosanthin on demethylation of p27 in HeLa cells / 中草药

ABSTRACT

Objective To investigate the effects of recombinant trichosanthin(rTCS) on methylation status and expression level of p27 gene in HeLa cells.Methods HeLa cells was treated by different concentration(20 ?g/mL,40 ?g/mL,and 80 ?g/mL) of rTCS for 48 h and then methylation-specific polymerase chain reaction(MSP) was used to detect the promoter methylation status of the p27 gene,real-time PCR was used to detect levels of p27 and DNMT1 mRNA,and Western blotting assay was used to detect expression level of p27 protein before and after treatment with rTCS.Results Low expression level and promoter methylation status of the p27 gene were detected in HeLa cells.Treatment with 40 ?g/mL rTCS totally demethylated p27 promoter.Treatment with 20 ?g/mL,40 ?g/mL or 80 ?g/mL rTCS resulted in a 2.22-,4.00-or 6.03-folds increase in p27 mRNA level,respectively,and also a great increase in p27 protein level.A high DNMT1 expression level was observed in HeLa cells and treatment with 40 ?g/mL rTCS resulted in a 78% decrease at the DNMT1 mRNA expression.Conclusion rTCS could reverse promoter hypermethylation and re-activate the expression of p27 gene by inhibiting DNMT1 expression in HeLa cells,which indicates its potential use in cancer therapy.