The Effects Of 5-Aza-CdR On The DBCCR1 Promoter Methylation In Human Bladder Cancer Lines T24 Cells

Objective: To explore the effect of 5-Aza-Cd R on the DBCCR1 gene promoter methylation status of human bladder cancer line T24 cells. To explore the effect of 5-Aza-Cd R on the DBCCR1 m RNA expression level of human bladder cancer line T24 cells.To explore the effect of DNA methyltransferase inhibitor 5-Aza-2 ’-deoxycytidine(5-Aza-Cd R) on the apoptosis and the proliferation of human bladder cancer line T24 cells.Methods: 1. The T24 cell culture and treatment: The human bladder cancer cell line T24 cells were cultured in RPIM 1640 medium. After subcultured the T24 cells, they were treated with containing different concentrations(0, 1, 4, 16, 64 umol/l) of methyltransferase inhibitor 5-Aza-Cd R with RPIM 1640 medium for 48 hours. 2. The DBCCR1 gene promoter methylation status of the drug treatment before and after of 24 cells were detected by the methylation specific PCR(MSP). 3. The DBCCR1 m RNA expression of human bladder cancer T24 cells which were cultured with different concentrations(0, 1, 4, 16, 64 umol/l) 5-Aza-Cd R for 48 hours were detected by Real-time fluorescence quantitative reverse transcription PCR(RFQ-RT-PCR). 4. The proliferation of human bladder cancer T24 cells which were cultured with different concentrations 5-Aza-Cd R for 48 hours was detected by using a MTS assay. 5. The apoptosis of T24 cells which were cultured with containing different concentrations(0, 1, 4, 16, 64 umol/l) of 5-Aza-Cd R for 48 hours were detected with flow cytometry.Results: 1. The result of DBCCR1 promoter methylation status of bladder cancer cell by MSP assay: The methylation status of the promoter of DBCCR1 gene in human bladder cancer T24 cell with normal cultivation condition showed completely methylation. When given 5-Aza-Cd R(16umol/l) in T24 cell for 48 hours, the methylated amplification band was negative and the non-methylated amplification band was positive. It suggested that the promoter of DBCCR1 gene in the human bladder cancer cell line T24 cells was hypermethylation and 5-Aza-Cd R could reverse the hypermethylation state of DBCCR1 promoter into non-methylation state. 2. Comparedwith control group, the DBCCR1 gene m RNA expression with different concentrations 5-Aza-Cd R groups had significant differences(P<0.01). As the drug concentration increasing, the m RNA expression level of DBCCR1 gene was growing. There were significant differences between any two groups(Welch test F = 4.288, P < 0.01, and pairwise comparison Dunnett T3 test, P values < 0.05). 3. The 5-Aza-Cd R had a obvious inhibition to the proliferation of T24 cells. Compared with the control group(without 5-Aza-Cd R), drug treatment groups had statistical significance(P<0.05). 4. The apoptosis of human bladder cancer T24 cells which were treated by different concentration of 5-Aza-Cd R for 48 hours were detected by flow cytometry(FITC Annexin Ⅴ/PI double staining) and the significant apoptosis cells were found. Compared with control group, the apoptosis of the medication groups increased with the increase of drug concentration. The difference of the apoptosis rates in any two groups was statistically significant(P<0.01).Conclusion: The prompter methylation status of human bladder cancer cell line T24 cells DBCCR1 gene could be reversed by DNA methyltransferase inhibitor 5-Aza-Cd R. 5-Aza-Cd R could enhance the expression level of DBCCR1 m RNA, inhibit the proliferation and induce the apoptosis of bladder cancer cell line T24 cells. That might provide a new research direction for the biological treatment of bladder cancer.