【摘要】 目的研究肿瘤抑制基因SLC5A8和 TMS1/ASC 在胶质瘤中的启动子区甲基化和mRNA 表达情况,探讨其与临床情况之间的关系。方法通过甲基化聚合酶链反应(MSP)检测SLC5A8和 TMS1/ASC 基因在88例胶质瘤组织、10例正常脑组织及胶质瘤细胞株 U251和 SHG-44中的 DNA 甲基化情况;通过传统逆转录聚合酶链反应(RT-PCR)和实时定量 PCR 检测 SLC5A8和TMS1/ASC 的 mRNA 表达情况;检测去甲基化试剂5-氮杂脱氧胞苷(5-Aza-CdR)作用于细胞株后,对基因 DNA 甲基化和 mRNA 表达的影响。结果在88例胶质瘤中,SLC5A8启动子区的甲基化发生率为70.45%(62/88),其甲基化率随胶质瘤恶性程度的增加而增高;TMS1/ASC 启动子区的甲基化发生率为51/88(57.95%),其甲基化率随胶质瘤恶性程度的增加而降低。10例正常脑组织中均未检测到 SLC5A8和 TMS1/ASC 基因的甲基化。在各级别胶质瘤中 SLC5A8和 TMS1/ASC 的 mRNA 与正常脑组织相比均表达下凋,差异均有统计学意义(均 P<0.05)。在 U251和 SHG-44胶质瘤细胞株中均检测到 SLC5A8和 TMS1/ASC 基因的甲基化,去甲基化试剂5-Aza-CdR 均能重新激活 SLC5A8和TMS1/ASC 基因的表达。结论 SLC5A8和 TMS1/ASC 基因在胶质瘤中的启动子区甲基化导致其mRNA 表达下调,其甲基化的发生率及 mRNA 表达与胶质瘤的恶性程度密切相关。更多还原

【Abstract】 Objective To study the methylation status of the SLC5A8 and TMS1/ASC genes, candidate tumor-inhibiting genes closely related to the central nervous system,in the promoter regions,the mRNA expression of these 2 genes,and their correlation with the clinical characteristics in human glioma. Methods The methylation status of SLC5A8 and TMS1/ASC genes in the promoter regions was studied by methylation specific PCR(MSP)in the specimens of primary astrocytoma from 88 patients,55 males and 33 females,aged 12-81,and 10 specimens of normal brain tissue,all obtained during operation,and in the human glioma cells of the lines U251 and SHG-44.The mRNA expression levels of SLCSA8 and TMS1/ASC genes in 30 specimens of primary glioma and 10 specimens of normal brain tissue were determined by conventional RT-PCR and real-time PCR.5-Aza-2′-deoxycytidine(5-Aza-CdR),a demethylating agent, was added into the culture fluid of the U251 and SHG-44 cells,and then real-time PCR was used to the methylation status and mRNA expression levels of the SLC5A8 and TMS1/ASC genes.Results MSP showed that the SLC5A8 promoter region was hypermethylated in 62 of the 88 specimens of astrocytoma (70.45%)and the TMS1/ASC promoter region was hypermethylated in 51 of the88 specimens of astrocytoma(57.95%).But no methylation of SLC5A8 and TMS1/ASC promoter was detected in the 10 specimens of normal brain tissue.The mRNA expression of SLC5A8 gene and the mRNA expression of TMS1/ASC gene in the specimens of astrocytoma of different pathological grades were all significantly decreased compared to the specimens of normal brain tissue(all P<0.05).The mRNA expression of SLC5A8 gene was not significantly related to the age and sex,however,the mRNA expression of TMS1/ASC was significantly higher in the age group>60 than in other age groups(all P<0.05).Both U251 and SHG-44 glioma cells showed methylation of SLC5A8 and TMS1/ASC genes and after the treatment of 5-Aza- CdR both genes showed reactivated mRNA expression.Conclusion Hypermethylation of SLC5A8 and TMS1/ASC genes in the promoter regions may play an important role in the down-regulation of their mRNA levels in glioma.The methylation frequency and mRNA levels of SLCSA8 or TMS1/ASC genes are closely related to the malignant development of glioma.更多还原