5-azacytidine induces PD-1 gene promoter demethylation and PD-1 protein expression in human lymphoid cell series Molt-4 cells

Objective To investigate the demethylation and changes in gene expression of programmed death receptor-1 （ PD-1） caused by methylation inhibitor 5- azacytidine （5-Zac） in lymphocyte series Molt-4 cells and its mechanism. Methods Molt-4 cells were cultured in different concentrations of 5-Zac（0, 5, 10 Umol/L）for 72 h, ratio of cell expressing PD-1 and apoptosis rate were detected by FCM, transcription of PD-1 gene mRNA was detected by RT-PCR. Molt-4 cell DNA of all groups were disposed by sodium bisulfite, PD-1 gene promoter fragment binded with transcription factor Brn-2 was amplified by PCR,these amplification fragments were transformed into E. coli. Positive clones were selected by sequencing,methylation status of the fragments binded with transcription factor Brn-2 was examined. Results S-Zac could increase the PD-1 expression of Molt-4 cells. PD-1 expression rate in 0 μmol/L 5-Zac（ 1. 13%±0.01% ） treated cells was found more lower than that in both 5 μmol/L and 10 μmol/L 5-Zac treated cells （18. 96% ±1. 87% , 63. 09% ± 6. 25% , P 〈 0. 05 ） , and they showed concentration-dependent （P 〈0.01）. Cells apoptosis rate and PD-1 mRNA expression were also observed increased significantly with 5-Zac treating. Demethylation probability of CG points showed significant difference between transcription factor Brn-2 binding site and other four locations （P 〈 0.05 ）. Conclusion 5 -Zac inhibits cell grouth in human lymphoid cell series Molt-4 by inducing PD-1 gene expression and promoter demethylation. PD-1 gene promoter binding transcription factor Brn-2 fragment CG point demethylation may be one of the important mechanisms in 5-Zac treated Molt-4 cells. Key words: 5-azacytidine; PD-1 expression; Cell apoptosis; Demethylation