[5-aza-2'-deoxycytidine induces changes of histone H3-lysine 9 methylation in bladder tumor cells].

OBJECTIVE

To investigate the effect of DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) on the histone H3-lysine 9 methylation status and gene expression of RUNX3 in human bladder tumor cells.

METHODS

Human bladder tumor cells of the line EJ were cultured and treated with 5-Aza-CdR for 24 h. MTT test was used to observe the proliferation and growth of the EJ cells. Other EJ cells were cultured and treated with 5-Aza-CdR and then chromatin immunoprecipitation assay was used to analyze the histone H3-lysine 9 methylation status of RUNX3 promoter and the second exon. The expression of RUNX3 was measured by RT-PCR.

RESULTS

The survival rate of the EJ cells treated with 5-Aza-CdR of the concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 micromol/L were 98.1%, 95.3%, 75.9%, 52.3%, 16.2%, and 7.7% respectively. And the survival rates of the EJ cells treated with 5-Aza-CdR for 12, 24, 36, 48, 72, and 96 hours were 89.4%, 85.2%, 78.6%, 37.1%, 8.9%, and 7.1% respectively. The survival rates of the 1.0, 2.0, 5.0, and 10.0 micromol/L group were significantly lower than that of the control group (all P < 0.05). Before the intervention, the amplified bands of the histone H3-lysine 9 methylation status of RUNX3 gene promoter and the second exon zone were obvious, but both disappeared after the treatment with 2.0 and 5.0 micromol/L 5-Aza-CdR. Before the intervention, RUNX3 gene was not expressed, and was expressed after treatment with 2.0 micromol/L 5-Aza-CdR.

CONCLUSION

5-Aza-CdR not only obviously inhibits the proliferation of human bladder cancer cells but also reactivates RUNX3 gene through demethylation of histone H3-lysine9 at RUNX3 promoter and the second exon. H3-lysine 9 trimethylation may be one of the most important reasons for gene inactivation of the RUNX3 gene.