Competing targets of microRNA-608 affect anxiety and hypertension

In HEK293T cells there is a 3-fold repression of the luciferase reporter by miR-608 (Fig 1F), whereas a minor effect is observed at the level of AChE activity in U937 cells.What happens at the AChE protein levels in U937 cells?"We found this comment important and performed an immunoblot analysis for the AChE protein in U937 cells exposed to miR-608 compared to controls.A robust effect was observed at the level of the AChE protein, and, as we previously showed, this effect was considerably larger than that observed in the catalytic activity of the AChE enzyme, compatible with a large fraction of the AChE protein being catalytically inactive (as we previously reported in Shaked et al.Immunity 2009).Fig 1G in the manuscript had been revised accordingly, demonstrating a similarly robust suppression of the AChE protein by miR-132 and miR-608."The authors argue that the reduced interaction between miR-608 and AChE in the presence of the minor allele potentiates the repression of other miR-608 targets.However the Western-blot shown in Fig. 3B does not corroborate this conclusion/claim."To refine the presentation we replaced the presented blot with a more representative one.We hope that the outcome of greater difference between the control and the miR-608 treated cells for the major and minor alleles will be clearer in this revised figure ."Moreover, for the in vitro experiments the authors restrict their analysis to CDC42 (Fig 3B andC), while arguing for the same effect in other targets (e.g.IL-6).It is essential to check the effect for additional miR-608 targets.Along this line, it would be most interesting to use a genome-wide approach to look at the overall targets of miR-608."We found this comment of utmost importance for understanding the complexity aspect of miR-608 effects.To address this issue more globally, we used the miRwalk database (http://www.umm.uniheidelberg.de/apps/zmf/mirwalk/)to identify the top 30 predicted targets of miR-608, searched for those targets that are expressed in HEK293T cells (which incidentally do not express IL6) and designed RT-PCR primers for all of these predicted targets(Supplementary Table 2).We then performed a microfluidics dynamic array experiment (Fluidigm http://www.