Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype
Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype
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DOI：
10.1073/pnas.0501475102
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发表时间：
2005-05-17
影响因子：
11.1
通讯作者：
Hayes, JD
中科院分区：
文献类型：
--
作者：
Devling, TWP;Lindsay, CD;Hayes, JD
描述了一种针对人Keap1的双链21核苷酸小干扰RNA(SiRNA)，它代表了一类独特的癌症化学预防药物。该siRNA可下调Keap1基因的表达，从而减轻核因子红系2 P45相关因子2(Nrf2)介导的基因表达的负调控。在人HaCaT角蛋白细胞中，siRNA在24~72 h内将内源性Keap1的mRNA水平降低到对照水平的30%，并能阻断标志标记的人Keap1蛋白的异位表达，但不能阻断V5标记的异位小鼠Keap1蛋白的异位表达。转染人HaCaT细胞的Keap1 siRNA显著提高了核因子红系2 P45相关因子2(Nrf2)蛋白的内源性水平，并使抗氧化反应元件驱动的报告基因的转录增加了2.3倍。此外，转染Keap1 siRNA 48h后，醛酮还原酶1c1/2和谷氨酸半胱氨酸连接酶催化和修饰亚基的表达增加了5-14倍。NAD(P)H：苯醌氧化还原酶1的活性也略有增加，为原来的3倍。转染后48h，Keap1 siRNA使细胞内谷胱甘肽含量增加1.75倍。因此，siRNA对Keap1的拮抗作用可以用于使人类细胞预先适应氧化应激，而不需要将它们暴露在氧化还原应激源中。
A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to < 30% of control levels between 24 and 72 h after transfection in human HaCaT keratin-ocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors.