胶质瘤细胞GDNF基因启动子Ⅰ区甲基化水平对其基因转录的影响

Effect of methylation levels of GDNF gene promoter Ⅰ region on its transcription in glioma U251 cells

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 explore the effect of methylation levels of glia cell line-derived neurotroplic factor （GDNF） promoter I region on its transcription in glioma U251 cells. Methods The methylation levels of GDNF promoter I region were detected before and after intervention with different concentrations of 5-aza-CR in glioma U251 cells. The GDNF mRNA expression was detected by RT-PCR in each group. Results The methylation levels of GDNF gene promoter I region declined after the intervention with 5-aza-cR compared to the control group. The methylation levels of GDNF gene promoter I region were significantly lower in 5 μmol, 10 p, mol and 20μmol 5-aza-CR intervention groups than that in the control group （P〈0.05）. The level of GDNF mRNA expression in U251 cells was significantly higher in the 5μmol 5-aza2CR intervention group than those in the control group and 1μmol, 10 μmol and 20 p, mol intervention groups （P〈0.05）. There were insignificant differences in the levels of GDNF mRNA expression in U251 cells among the control group and 1μmol, 10μmol and 20μmol 5-aza-CR intervention groups （P〉0.05）. Conclusion The demethylation of GDNF gene promoter [ region, which is produced by 5-aza-CR, may influence the expression of GDNF in U251 cell.