Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells
Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells
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DOI：
10.1038/sj.onc.1210098
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发表时间：
2007-05-03
期刊：
影响因子：
8
通讯作者：
Ionov, Y.
中科院分区：
文献类型：
--
作者：
Ivanov, I.;Lo, K. C.;Ionov, Y.
抑制细胞中无义介导的衰变（NMD）机制导致携带过早翻译终止密码子的转录本稳定。一种被称为NMD抑制基因鉴定（GINI）的策略被提出来鉴定携带无义突变的基因。结肠癌细胞系中含有移码突变的基因已被使用改良版的GINI识别出来。为了提高使用GINI识别突变基因的效率，我们现在进一步改进了该策略。在这种方法中，用艾美汀抑制NMD的同时，用咖啡因阻断hUpf1蛋白的磷酸化，从而抑制NMD。此外，为了增强GINI策略，比较咖啡因预处理后单独抑制转录或抑制转录与NMD一起产生的mRNA水平变化，以有效识别由于NMD抑制的应激反应而产生的假阳性。为了证明这种方法提高了效率，我们分析了显示微卫星不稳定性的结肠癌细胞系。在FXR1、SEC31L1、NCOR1、BAT3、PHF14、ZNF294、C19ORF5基因以及功能未知的蛋白编码基因中发现双等位基因失活突变。
Inhibition of the nonsense-mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene identification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations. Genes containing frameshift mutations in colon cancer cell line have been identified using a modified version of GINI. To increase the efficiency of identifying mutant genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhibiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analysed colon cancer cell lines showing microsatellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC31L1, NCOR1, BAT3, PHF14, ZNF294, C19ORF5 genes as well as genes coding for proteins with yet unknown functions.