Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2.
Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2.
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DOI：
10.1186/1758-907x-2-3
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发表时间：
2011-03-14
期刊：
影响因子：
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通讯作者：
Sander C
中科院分区：
文献类型：
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作者：
Schultz N;Marenstein DR;De Angelis DA;Wang WQ;Nelander S;Jacobsen A;Marks DS;Massagué J;Sander C
RNA干扰（RNAi）筛选已被用于识别各种生物信号转导途径的新成分。我们对人角质形成细胞系中转化生长因子(TGF)-β通路的新成员进行了小干扰（si）RNA筛选。TGF-β通路是哺乳动物细胞增殖和存活不可或缺的一部分，异常的TGF-β反应与癌症密切相关。我们分析了针对6000个基因的单个sirna对绿色荧光蛋白(GFP)-SMAD2报告融合蛋白核易位的影响。令人惊讶的是，我们没有发现新的TGF-β通路成员，但我们确实发现了主要的脱靶效应。所有siRNA的撞击，无论其预期的直接目标是什么，都通过微（mi） rna样脱靶效应降低了两种已知上游途径成分TGF-β受体1和2 （TGFBR1和TGFBR2）的mRNA水平。这些脱靶效应的规模是显著的，在无偏siRNA文库中至少有1%的序列对这两个基因中的一个具有可测量的脱靶效应。如果通路输出对特定通路成分的水平非常剂量敏感，那么通过脱靶效应相对较小的信息水平降低似乎可以对测定产生主要影响。为了寻找机制细节，我们确定了与脱靶效应相关的多个mirna样序列特征。基于这些结果，我们确定了miR-20a、miR-34a和miR-373是抑制TGFBR2表达的mirna。我们的研究结果指出了miRNA/siRNA靶标预测方法的潜在改进，并表明II型TGF-β受体受到多种miRNA的调节。我们还得出结论，使用单分析读数的大型文库在siRNA筛选中获得误导性结果的风险很大。控制和拯救实验在解释这些筛选中是必不可少的，改进减少或预测RNAi脱靶效应的方法将是有益的。
RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer. We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-β pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression. Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial.