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系统分析 RNAi 筛选中的脱靶效应揭示了影响 TRAIL 诱导细胞凋亡敏感性的 microRNAs。

Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis.

机构信息

Work performed at: Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

出版信息

BMC Genomics. 2010 Mar 15;11:175. doi: 10.1186/1471-2164-11-175.

Abstract

BACKGROUND

RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs), causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s).

RESULTS

We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects) is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death.

CONCLUSIONS

We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.

摘要

背景

siRNA 对 RNA 的抑制作用是一种常用于鉴定特定生物过程所需基因的方法。然而,siRNA 的 RNAi 筛选受到 siRNA 的非特异性或脱靶效应的阻碍,使得很难将真正的命中与假阳性区分开来。人们认为,RNAi 实验中观察到的许多脱靶效应是由于 siRNA 充当 microRNAs (miRNAs),通过与 6 或 7 nt“种子”序列的匹配,导致非预期靶基因的表达减少。我们在 TRAIL 凋亡诱导途径的新型调节剂的 siRNA 筛选过程中仔细检查了脱靶效应。

结果

我们在 TRAIL 凋亡筛选的前 20 个 siRNA 中发现了 3 个六聚体和 3 个七聚体种子序列多次出现。使用一种新的统计富集方法,我们系统地鉴定了另外 17 个六聚体和 13 个七聚体种子序列在高得分 siRNA 中富集。这些种子序列之一的存在(可以解释 8 个已确认的脱靶效应中的 6 个)足以引起表型。其中三个种子序列出现在人类 miRNA miR-26a、miR-145 和 miR-384 中。这些 miRNA 的模拟物的转染可保护几种细胞类型免受 TRAIL 诱导的细胞死亡。

结论

我们证明了 miR-26a、miR-145 和 miR-384 在 TRAIL 诱导的凋亡中的作用。此外,这些结果表明,RNAi 筛选富集了具有相关脱靶效应的 siRNA。高得分 siRNA 中某些种子序列的过度表达可以识别出其中一些效应,我们证明了对富集种子序列进行这种系统分析的有用性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fa44/2996961/aca87c134f5d/1471-2164-11-175-1.jpg

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