抑制突变TP53增强胶质母细胞瘤细胞替莫唑胺敏感性及其相关机制  

Down Regulating Mutant TP53 Enhances Temozolomide Sensitivity in Glioblastoma cells

作　　者：王翔[1] 陈锦秀[2] 毛庆[1] 刘艳辉[1] 游潮[1] 

机构地区：[1]四川大学华西医院神经外科,四川成都610041 [2]四川省肿瘤医院,四川成都610041

出　　处：《中国神经肿瘤杂志》2011年第3期152-159,共8页Chinese Journal of Neuro-Oncology

摘　　要：背景与目的：胶质瘤化疗耐药是胶质瘤治疗效果差的一个瓶颈。导致化疗耐药的分子机制目前尚未完全清楚。突变TP53的＂获得功能＂被认为是决定肿瘤发生及发展中的重要因素。本研究目的在于探讨突变TP53＂获得功能＂在多形性胶质母细胞瘤化疗耐药中的作用及其相关机制。方法：对人脑胶质瘤母细胞株T98G细胞进行mRNA测序,确定其TP53基因是否突变及突变位点。采用脂质体法包裹化学合成的突变TP53干扰小RNA片段（siRNA）转染T98G细胞。运用RT-PCR方法在转染后的第1天至第5天检测肿瘤中突变TP53在mRNA水平的表达,及相应时点6-氧甲基鸟嘌呤-DNA甲基转移酶（MGMT）mRNA的表达。将转染siRNA的T98G细胞接种于96孔板中,加入梯度浓度（10-1000μmol/L）的替莫唑胺。模拟人体用药,替莫唑胺组在头5天内每天更换培养基,加入替莫唑胺;对照组加入司莫司汀。用MTT法观察各组在突变TP53沉默前后的化疗敏感性变化。结果：T98G细胞的237号密码子发生突变,且高表达突变TP53及MGMT mRNA。设计的siRNA可以有效地沉默T98G细胞株内突变TP53基因,使其在mRNA水平不表达或者低表达,沉默作用时间可达5天。沉默突变TP53基因后,T98G细胞株对替莫唑胺的敏感性增加4倍,对司莫司汀的敏感性无明显改变。沉默突变TP53基因后,可使MGMT的表达降低,两者呈正相关。结论：突变型TP53能增强胶质母细胞瘤细胞对替莫唑胺化疗的耐药性,其耐药机制可能是通过上调MGMT的表达实现的。BACKGROUND OBJECTIVE： The drug resistance is one of the major obstacles in cancer chemotherapy.The molecular mechanism leading to drug resistance is still not fully understood.The ＂gain of function＂ of mutant TP53 is an important determinant in human tumor development and progression.This study aimed to investigate the possible mechanism of mutant TP53 inducing temozolomide resistance in glioblastoma cells.METHODS： Synthetic small interfering RNA was used to knock down mutant TP53 in T98G cell line,a human glioblastoma cell line with a mutation at codon 237 of gene TP53,which was proved by DNA sequencing.The expression of mutant TP53 mRNA was detected ＂silenced＂ by reverse transcriptase-polymerase chain reaction（RT-PCR） in consecutive 5 days.Viable cell survival was measured to detect the sensitivity when these cells were exposed to temozolomide（10~1000μmol/L）.The cell culture medium was removed and temozolomide was added every day at the early 5 days.The O6-methylguanine DNA-methyltransferase（MGMT）expression at mRNA level was analyzed after the mutant TP53 knockdown as well.RESULTS：Both mutant TP53 and MGMT showed a strong expression in T98G cells.The knockdown efficiency of mutant TP53 with small interfering RNA from the first day to fifth day was 83%,95%,93%,76%,and 72% respectively.Mutant TP53 knockdown led to a fivefold increase in the sensitivity of T98G cells to temozolomide in the cell experiment.In addition,mutant TP53 knockdown induced the down-regulation of MGMT expression.CONCLUSION：Down regulating mutant TP53 could enhance the chemosensitivity of malignant gliomas to temozolomide,which could be through decrease MGMT expression.

关 键 词：胶质母细胞瘤 突变 6-氧甲基鸟嘌呤-DNA甲基转移酶 肿瘤抑癌基因TP53 RNA干扰 

分 类 号：R739.41[医药卫生—肿瘤]