脑胶质瘤中SLC5A8和TMS1/ASC基因启动子区甲基化及mRNA表达的研究

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SLCSA8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SLCSA8和TMS1／ASC的mRNA与正常脑组织相比均表达下调,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。

The methylation and mRNA expression of SLC5A8 and TMS1/ASC genes in human glioma

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 SLC5A8 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 SLC5A8 or TMS1/ASC genes are closely related to the malignant development of glioma.