Schultz Nikolaus, Marenstein Dina R, De Angelis Dino A, Wang Wei-Qing, Nelander Sven, Jacobsen Anders, Marks Debora S, Massagué Joan, Sander Chris
Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Silence. 2011 Mar 14;2:3. doi: 10.1186/1758-907X-2-3.
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.
RNA干扰（RNAi）筛选已被用于在多种生物体中鉴定信号转导通路的新组分。我们在人角质形成细胞系中针对转化生长因子（TGF）-β通路的新成员进行了小干扰（si）RNA筛选。TGF-β通路对哺乳动物细胞增殖和存活至关重要，且TGF-β反应异常与癌症密切相关。
我们检测了靶向6000个基因中每个基因的单个siRNA对绿色荧光蛋白（GFP）-SMAD2报告融合蛋白核转位的影响程度。令人惊讶的是，我们未发现新的TGF-β通路成员，但发现了显性脱靶效应。所有siRNA命中物，无论其预期的直接靶点是什么，都通过微小（mi）RNA样脱靶效应降低了两个已知上游通路组分——TGF-β受体1和2（TGFBR1和TGFBR2）的mRNA水平。这些脱靶效应的规模显著，无偏差siRNA文库中至少1%的序列对这两个基因之一具有可测量的脱靶效应。如果通路输出对特定通路组分的水平非常敏感，那么通过脱靶效应导致的相对较小的信使水平降低似乎可能对检测产生显性影响。为了寻找机制细节，我们鉴定了与脱靶效应相关的多个miRNA样序列特征。基于这些结果，我们鉴定出miR-20a、miR-34a和miR-373为抑制TGFBR2表达的miRNA。
我们的发现指出了miRNA/siRNA靶点预测方法的潜在改进方向，并表明II型TGF-β受体受多种miRNA调控。我们还得出结论，在使用具有单一检测读数的大型文库进行siRNA筛选时，获得误导性结果的风险很大。对照和挽救实验对于此类筛选的解释至关重要，改进方法以减少或预测RNAi脱靶效应将是有益的。
