神经胶质瘤中IDH2 R172G突变对基因表观修饰的研究

Genetic Epigenetic Modification of IDH2 R172G Mutation in Glioma

目的分析在神经胶质瘤细胞中异柠檬酸脱氢酶2(IDH2)突变后P15ink4b转录水平的变化,寻找和基因表观遗传修饰相关的异常表达的蛋白。重点关注P15ink4b启动子区甲基化水平变化,进而分析IDH2突变、肿瘤抑制基因P15ink4b、2-HG和甲基胞嘧啶羟化酶(TET)之间的关系。方法 p EGFP-N1、p EGFP-N1-IDH2M、p EGFP-N1-IDH2WT3个质粒转染C6细胞48h后,收获细胞Western Blot检测抑癌蛋白P15ink4b水平,q PCR检测P15ink4bmRNA变化,5-hm C检测试剂盒检测P15ink4b启动子区Cp G岛,CCGG位点5-hm C概率。结果 IDH2突变组抑癌蛋白P15ink4b表达明显低于对照组。IDH2突变组P15ink4bmRNA水平低于对照组,差异有统计学意义(P=0.043)。随着2-羟基戊二酸浓度加大,细胞活性明显增强,差异有统计学意义(P<0.05)。p EGFP-N1-IDH2WT组8个CCGG位点第二个C的5-hm C的概率都大于80%,而p EGFP-N1-IDH2M组都小于1%。结论 IDH2突变后产生的2-羟基戊二酸可以抑制TET酶活,从而去甲基化程度降低,使得抑癌基因P15ink4b启动子区甲基化程度增强,抑制P15ink4b基因表达,促进细胞增殖迁移。

Objective To analyze the level of P15^ink4 bmRNA after isocitrate dehydrogenase 2（ IDH2）mutation in glioma cells and search for the protein abnormal expression of epigenetic modifications of gene.The changes of methylation level in P15ink4 bpromoter region were focused and the relationship were analyzed among IDH2 mutations,tumor suppressor gene P15ink4 b,2-HG and translocation methylcytosine dioxygenase（ TET）. Methods After three plasmids of p EGFP-N1,p EGFP-N1-IDH2 M,p EGFP-N1-IDH2 WTwere transfected into C6 cells for 48 h,all the cells were harvested. The level of suppression cancer protein P15ink4 b were detected by western blot,P15^ink4 bmRNA by q PCR,and the 5-hm C probability in CCGG sites of Cp G island of P15^ink4 bpromoter region by 5-hm C test kits. Results The 5-hm C expression of P15^ink4 bin IDH2mutation group was significantly lower than that of the control group,and the difference was statistically significant（ P = 0. 043）. The cell activity was increased with the level of 2-hydroxyglutarate concentration,and the difference was statistically significant（ P〈0. 05）. All the probability of the second C in eight CCGG sites of p EGFP-N1-IDH2 WTgroup was higher than 80%,but less than 1% in the p EGFP-N1-IDH2 M group.Conclusion The 2-hydroxyglutarate produced by IDH2 mutation can inhibit the activity of TET enzyme,decrease the demethylation level in anti-oncogene P15^ink4 bpromoter region,finally inhibit the expression of P15ink4 bgene,and promote cell proliferation and migration.