Abstract:

Objective To investigate the methylation, expression, and transcription of the promoter of reduced folate carrier 1 (RFC1) gene of U937 cell line and to observe the effects of variations of the methylation level on the cell apoptosis and cycle induced by methotrexate (MTX). Methods The quantitative real-time methylation-specific PCR and real-time quantitative PCR were adopted to detect the methylation status and mRNA expression of RFC1 gene promoter of U937 cell line after being incubated by 5-azacytidine. The effects of variations of RFC1 gene expression on the apoptosis and cell cycle of U937 cells induced by MTX were verified by the flow cytometry. Results The un-methylation rate of RFC1 gene promoter of U937 cells was (8.09±0.87)% which was in high methylation status and increased to (9.44±2.01)%, (13.51±2.19)%, and (15.76±1.65)% after being demethylated by 5-azacytidine of 1, 2, and 4 μmol/L for 72 h, respectively. Compared with the control group, the un-methylation rate of RFC1 promoter significantly increased (P=0.005, P=0.001) and the mRNA expression of RFC1 promoter increased (P=0.003, P=0.000) after being treated by 5-azacytidine of 2 and 4 μmol/L. And 5-azacytidine of 2 μmol/L inhibited the S phase and increased G1 phase of cell cycle. MTX of 5 nmol/L induced the apoptosis and inhibited the S phase of cell cycle. The combination of MTX and 5-azacytidine significantly increased the apoptosis and inhibited S phase of cell cycle. Conclusion The methylation level of RFC1 gene promoter of U937 cell line is abnormally high. And 5-azacytidine can demethylate and increase the mRNA expression of RFC1 gene promoter, therefore enhance the apoptosis effect of MTX and specifically inhibit the S phase of cell cycle.

Key words: U937, DNA methylation, reduced folate carrier gene, 5-azacytidine, methotrexate