5-杂氮胞苷诱导人淋巴细胞系Molt4细胞PD-1基因启动子去甲基化及PD-1蛋白表达的影响

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

目的 以T淋巴细胞株Molt-4细胞为模型,探讨甲基化抑制剂5-杂氮胞苷（5-Zac）对淋巴细胞表面程序性死亡受体-1（PD-1）基因肩动子的去甲基化作用及其诱导的PD-1基因表达的改变,并进一步研究去甲基化作用与PD-1基因表达之间的关系.方法 以不同浓度的5-Zac（0、5、10μmoL/L）作用于体外培养的Molt-4细胞72 h,流式细胞术（FCM）检测细胞表面表达PD-1的Molt-4细胞比例和细胞凋亡率;反转录-聚合酶链反应（RT-PCR）检测5-Zac作用后PD-1基因mRNA的转录水平;亚硫酸氧钠处理各组Molt-4细胞DNA,PCR扩增与转录因子Brn-2结合的PD-1启动子基因片段,转化感受态大肠杆菌,挑克隆测序,检测扩增的PD-1启动子片段甲基化状态.结果 0、5、10μmol/L的5-Zac作用于Molt-4细胞72 h后,PD-1在细胞表面的表达率分别为1.13%4±0.01%、18.96%±1.87%、63.09%±6.25%,并呈现浓度依赖性（P〈0.05）;PD-1基因mRNA表达量显著增加;细胞凋亡检测结果显示与未处理组相比,5-Zac处理72 h后Molt-4细胞的凋亡率显著增加,3组凋亡率分别为1.9%4±0.06%、8.98%4±1.36%、24.5%4±3.68%,差异有统计学意义（P〈0.01）;3组DNA亚硫酸氢钠测序结果表明,5-Zac处理后,与转录因子Bin-2结合位置的CG点明显受到去基化影响,其去甲基化概率与周围其他CG点去甲基化概率比较差异有统计学意义（P〈0.05）.结论 甲基化抑制剂5-Zac可导致体外培养的淋巴细胞系Molt-4细胞表面PD-1表达显著增加,PD-1基因mRNA表达增加,细胞凋亡率增高;PD-1基因活化和表达的增加可能与5-Zac降低PD-1基因启动子上与转录因子Bin-2结合位置的CG点甲基化水平,从而促进Brn-2与基因启动子结合,使转录表达增加有关.

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.