DNA甲基化在脑胶质瘤中调控囊泡相关膜蛋白8基因的表达

DNA methylation regulated VAMP8 expression in glioma

目的探讨囊泡相关膜蛋白8(VAMP8)基因在脑胶质瘤中的表达及DNA甲基化对其的影响。方法通过荧光定量PCR分析VAMP8基因在脑胶质瘤中的表达水平;使用亚硫酸氢盐测序PCR检测VAMP8基因启动子甲基化程度;通过DNA甲基转移酶抑制剂处理脑胶质瘤细胞,检测DNA甲基化程度对VAMP8基因表达的影响;通过双荧光素酶报告基因实验验证DNA甲基化程度对VAMP8基因启动子活性的影响。结果与正常脑组织相比,VAMP8基因在脑胶质瘤组织中高表达(P=0.002),其表达水平与DNA甲基化程度相关(相关系数r=-0.533,P=5.8×10-5);脑胶质瘤组织中VAMP8基因转录起始区上游6个Cp G位点甲基化程度低(P<0.05)。VAMP8基因在脑胶质瘤细胞中的表达量受DNA甲基化水平的调控,处于高甲基化状态的VAMP8基因启动子活性较低(P=0.028)。结论脑胶质瘤组织DNA甲基化程度较低是VAMP8基因高表达的原因之一。

Objective To investigate the expression level of vesicle associated membrane protein 8 （VAMP8） in glioma and the effect of DNA methylation on it. Methods The expression of VAMP8 in glioam was detect- ed by real-time fluorescence quantitative PCR. The DNA methylation level in VAMP8 promoter was explore via bisulfate sequencing PCR sequence. Glioma cell lines were exposed to DNA methyltransferase inhibitor in vitro to observe the influence of DNA methylation level on VAMP8 expression. The effect of DNA methyl- ation on VAMP8 promoter activity was discovered by Dual-Luciferase assay. Results VAMP8 was highly expressed in glioma （P=0.002） and its expression level was correlated with DNA methylation （r=-0.533, P=5.8×10^-5）. The methylation level of 6 CpG sites in the upstream of VAMP8 transcription initiation region in glioma was significantly lower than that in normal brain tissue （P 〈 0.05）. The expression of VAMP8 was regulated by DNA methylation level in glioma cell lines. The promoter activity of VAMP8 was lower in the high DNA methylation state （P=0.028）. Conclusion The low DNA methylation level was one of the reasons of the high expression of VAMP8 in glioma.