Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2

Abstract

BackgroundRNA 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.ResultsWe 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.ConclusionsOur 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.

Altered axonal targeting and short-term plasticity in the hippocampus of Disc1 mutant mice

“…Targeting of a small portion of cells within the brain cannot reproduce the timing and magnitude of a germ-line genetic disruption and it may result in unpredictable interactions with the WT context surrounding them. Another likely explanation for some of these discrepancies is off-target effects, a well-known confounding factor of RNAi-based approaches (59,60). Overall, our findings suggest that results from studies using RNAi techniques may need to be reevaluated in carefully designed animal models.…”

MicroRNA-520/373 family functions as a tumor suppressor in estrogen receptor negative breast cancer by targeting NF-κB and TGF-β signaling pathways

“…Thus, our data support a contextdependent effect of miR-520/373 on patient outcome. In a recent study that aimed at the identification of genes regulating TGF-b signaling in HaCaT keratinocytes, miR-373 was coincidentally identified as a regulator of TGFBR2 (Schultz et al, 2011). This finding demonstrates that the regulation of TGF-b signaling via miR-520/373 is not restricted to breast cancer and that further studies are thus needed to better understand this complex regulation.…”

Induction of Specific MicroRNAs Inhibits Cutaneous Wound Healing

“…Interestingly, mRNA levels of TGF␤RII were not significantly altered in contrast to the marked downregulation at the protein level, suggesting the potential role of miRNAs in translational inhibition. Indeed, TGF␤RII has been recently confirmed as target for miR-20a in human keratinocytes (48), suggesting that induction of this miRNA in VUs can be responsible for suppression of TGF␤ signaling. Further studies are needed to confirm the role of miR-20a in wound healing; however, induction of miR-20a can potentially provide the explanation for limited success of TGF␤ in clinical trials, in addition to promising animal studies (7).…”