Study of 5-Aza-CdR on transcription regulation of RASSF1A gene in the BIU87 cell line.

OBJECTIVES

To investigate the effect of 5-Aza-CdR on methylation and expression of RASSF1A gene in the human bladder cancer BIU87 cell line.

METHODS

Cultured BIU87 cells were treated with increasing concentrations (0.1, 0.5, 1.0, 5.0 micromol/l) of 5-Aza-CdR. MTT was used to detect the proliferation of BIU87 cells. The methylation status and expression level of RASSF1A gene in BIU87 cells were analyzed by methylation-specific polymerase chain reaction and RT-PCR before and after treatment with 5-Aza-CdR. Meanwhile, Western blot was used to detect the changes of RASSF1A protein.

RESULTS

In this study we found that the growth velocity of the BIU87 cell line was suppressed to various degrees after having been treated with different concentrations of 5-Aza-CdR, and the survival rate decreased with increasing 5- Aza-CdR concentrations. Besides, the methylation status of RASSF1A was obviously reversed and mRNA was re-expressed after treatment with 5-Aza-CdR. Expression of RASSF1A protein increased with increasing 5-Aza-CdR concentrations.

CONCLUSIONS

Our results suggest that the tumor-suppressive effect of 5-Aza-CdR may result from reactivation of silenced RASSF1A through a demethylation function.