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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。
Objective To investigate the promoter methylation and mRNA expression of tumor suppressor genes SLC5A8 and TMS1 / ASC in gliomas and to explore their relationship with clinical conditions. Methods The DNA methylation status of SLC5A8 and TMS1 / ASC in 88 glioma tissues, 10 normal brain tissues and glioma cell lines U251 and SHG-44 was detected by methylation-based polymerase chain reaction (MSP) . The mRNA expression of SLC5A8 and TMS1 / ASC was detected by RT-PCR and real-time PCR. The effect of demethylation reagent 5-azacytidine (5-Aza-CdR) On cell DNA, on DNA methylation and mRNA expression. Results In 88 gliomas, the methylation rate of SLC5A8 promoter region was 70.45% (62/88). The methylation rate of SLC5A8 promoter region increased with the malignant degree of glioma. The promoter region of TMS1 / ASC promoter Methylation rate was 51/88 (57.95%), the methylation rate decreased with the increase of malignant glioma. No methylation of SLC5A8 and TMS1 / ASC genes was detected in 10 normal brain tissues. The mRNA expression of SLC5A8 and TMS1 / ASC in gliomas at all levels were significantly lower than that in normal brain tissues (all P <0.05). Methylation of both SLC5A8 and TMS1 / ASC genes was detected in both U251 and SHG-44 glioma cell lines, and demethylation reagent 5-Aza-CdR both reactivated the expression of SLC5A8 and TMS1 / ASC genes. Conclusion The methylation of SLC5A8 and TMS1 / ASC genes in glioma leads to the down-regulation of their mRNA expression. The incidence of methylation and the mRNA expression of SLC5A8 and TMS1 / ASC are closely related to the malignant degree of glioma.