Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors
Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors
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DOI：
10.1074/jbc.m409948200
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发表时间：
2005-01-07
影响因子：
4.8
通讯作者：
Taira, K
中科院分区：
文献类型：
--
作者：
Futami, T;Miyagishi, M;Taira, K
本文描述了针对数百个凋亡相关基因的小干扰RNA表达载体文库的构建，并将该文库应用于thapsigargin(TG)诱导的细胞凋亡的研究。Thapsigargin触发内质网应激，继而导致细胞凋亡，但这一过程背后的分子机制尚不完全清楚。利用我们的文库，我们发现了三个抗凋亡基因，即noxA、E2F1和MAPK1，以及在细胞凋亡途径中已鉴定的基因。与其他人的建议相反，我们的数据揭示：(I)TG诱导的细胞凋亡与Apaf1以caspase-3和caspase-9不依赖的方式相关；(Ii)E2F1-PUMA途径可能参与；(Iii)ERK途径通过MAP3K8(丝裂原激活的蛋白激酶8)是TG诱导细胞凋亡所必需的。我们的研究清楚地表明，我们的方法可以有效地识别意外和新的基因，并为使用小干扰RNA表达载体文库对信号网络和通路进行综合分析的有效性和实用性提供了证据。
We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.