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目的探讨胶质瘤细胞胶质细胞原性神经营养因子(GDNF)基因启动子Ⅰ区甲基化水平对其基因转录的影响。方法体外培养胶质瘤U251细胞,加入不同浓度5-氮杂胞苷(浓度分别为1、5、10和20μmol/L)干预,以加入PBS为对照。采用重亚硫酸盐测序法测定GDNF基因启动子Ⅰ区甲基化水平,RT-PCR检测GDNF m RNA的表达。结果与PBS组相比,1μmol/L 5-氮杂胞苷对GDNF基因启动子Ⅰ区甲基化水平无显著影响(P>0.05),5、10和20μmol/L 5-氮杂胞苷均显著降低其甲基化水平(P<0.05)。与PBS组相比,1μmol/L 5-氮杂胞苷对GDNF m RNA表达水平无显著影响(P>0.05),5μmol/L 5-氮杂胞苷显著增加其表达水平(P<0.05),但随着浓度进一步增加(10、20μmol/L),其表达水平逐渐降低。结论 5-氮杂胞苷对GDNF基因具有去甲基化作用;GDNF启动子Ⅰ区去甲基化能够增加GDNF基因的转录水平。
Objective To investigate the effect of methylation level of promoter region Ⅰ of glial cell line-derived neurotrophic factor (GDNF) gene on the gene transcription of glioma cells. Methods Glioma U251 cells were cultured in vitro and treated with various concentrations of 5-azacytidine (1, 5, 10 and 20 μmol / L). PBS was added as a control. The methylation level of GDNF gene promoter region Ⅰ was detected by bisulfite sequencing, and the expression of GDNF m RNA was detected by RT-PCR. Results Compared with PBS group, 1μmol / L 5-azacytidine had no significant effect on the methylation level of GDNF promoter Ⅰ (P> 0.05), while 5, 10 and 20μmol / L 5-azacytidine Significantly reduced its methylation level (P <0.05). Compared with PBS group, 1μmol / L 5-azacytidine had no significant effect on the expression of GDNF m RNA (P> 0.05), and 5μmol / L 5-azacytidine significantly increased its expression level (P <0.05) But with the further increase of concentration (10,20μmol / L), its expression level decreased gradually. Conclusion 5-azacytidine can demethylate GDNF gene and demethylation of GDNF promoter can increase the transcription level of GDNF gene.