Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference
Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference
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DOI：
10.1038/cgt.2010.35
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发表时间：
2010-11-01
影响因子：
6.4
通讯作者：
Nemunaitis, J.
中科院分区：
文献类型：
--
作者：
Rao, D. D.;Maples, P. B.;Nemunaitis, J.
RNA干扰（RNA interference，RNAi）是一种通过转录、转录后和翻译机制抑制基因表达的自然细胞调控过程。模拟这一过程的合成方法（小干扰RNA（siRNA），短发夹RNA（shRNA））已被证明在这方面同样有效。我们开发了一种新的“双功能”RNAi策略，进一步优化了靶基因敲除结果。针对Stathmin 1产生双功能构建体（bi-sh-STMN 1），Stathmin 1是在人类癌症中过表达的关键微管蛋白调节剂。假设双功能构建体同时抑制靶mRNA的翻译（不依赖于切割，mRNA封存和降解）并通过切割依赖性活动降解（通过RNase H样切割）转录后mRNA。Bi-sh-STMN 1在与靶向相同序列的常规shRNA和siRNA的平行比较中显示出增强的效力和耐久性。通过快速扩增互补DNA末端（RACE）分析显示，bi-sh-STMN 1增强的STMN 1蛋白敲除伴随着mRNA水平上的靶位点切割。Bi-sh-STMN 1在mRNA水平上也表现出与其多效应子沉默机制一致的敲低动力学。双功能shRNA是一种高效且有利的方法，其在显著低于常规shRNA或siRNA的浓度下介导RNAi。这些结果支持进一步的评价。Cancer Gene Therapy（2010）17，780-791; doi：10.1038/cgt.2010.35; 2010年7月2日在线发表
RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations. Cancer Gene Therapy (2010) 17, 780-791; doi:10.1038/cgt.2010.35; published online 2 July 2010