【摘要】 目的应用GINI筛选微卫星不稳定型大肠癌中新的突变基因。方法采用NMD抑制药物emetine(依米丁)联合actinomycin D(放线菌素D),分别处理高度微卫星不稳定型(MSI-H)大肠癌细胞株HCT116和微卫星稳定(MSS)的大肠癌细胞株HT29,以及对照的MSS乳腺癌细胞株,以全基因组cDNA芯片检测经药物处理和不经药物处理的细胞株的mRNA表达变化。经过芯片数据分析筛选在HCT116细胞中mRNA表达量特异性升高的基因,以DNA直接测序法检测突变,并在22个大肠癌细胞株及30例原发癌组织中验证。结果在MSI-H大肠癌细胞株HCT116中筛选出新的突变基因ARV1和ZNF294,并在8个MSI-H细胞株中验证其突变率分别为37.5%和87.5%,在20例MSI-H原发大肠癌组织中突变率均为35%。结论基于NMD抑制的新的基因筛选方法GINI,能够相对快速、有效的检出发生突变的基因。更多还原

【Abstract】 Objective To identify new mutated gene in microsatellite instable colorectal carcinoma through GINI. Methods Microsatellite instable-high(MSI-H) colon cancer cell line HCT116, microsatellite stable(MSS) colon cancer cell line HT29, as well as control MSS breast cancer cell lines were treated by NMD inhibition drug emetine and actinomycin D. The changes of mRNA expression of drug treated and untreated cell lines were detected with whole genomic cDNA microarray. Candidate genes with specifically increased mRNA expression in HCT116 were screened for mutation with direct DNA sequencing, and their mutations were identified in 22 colon cancer cell lines and 30 fresh colon cancer tissues. Results Two new mutated genes ARV1 and ZNF294 were screened out in MSI-H colon cancer cell line HCT116, and further validated in 37.5% and 87.5% of 8 MSI-H colon cancer cell lines respectively, as well as in 35% of 20 MSI-H primary tumors. Conclusions GINI-the new gene screening method which based on NMD inhibition, can be used to identify new mutated genes more rapidly and efficiently relatively.更多还原