In this study, we used a variety of bioinformatic, molecular and cell biological methods to demonstrate the role of miRNAs in the post-transcriptional control of utrophin expression. We show that at least six miRNAs target the utrophin 3′-UTR. We also demonstrate that inhibition of utrophin-targeting miRNAs can de-repress the utrophin 3′-UTR, leading to an upregulation of utrophin protein expression. We suggest that these mechanisms could be targeted to upregulate utrophin in DMD.
Previous work has shown that the utrophin 5′-UTR inhibits cap-dependent translation in muscle, probably due to its predicted complex secondary structure, but that an IRES located in the 5′-UTR is activated during muscle regeneration and in proliferating C2C12 myoblasts 
. In our study, we confirm the inhibitory effect of the utrophin 5′-UTR in C2C12 cells. Interestingly, we find that the 5′- and 3′-UTRs can act synergistically, such that each potentiates the inhibition caused by the other. Our ribosomal profiling experiments using reporter constructs shed light on the mechanisms of inhibition by the 5′- and 3′-UTRs, suggesting that they cause an inhibition of translational initiation from the utrophin-A mRNA, thus limiting ribosome occupancy.
Having shown the importance of the 3′-UTR in repressing utrophin translation, and its interaction with the 5′-UTR, we demonstrated that this repression is mediated, at least in part, by miRNAs. We identified five new miRNAs (let-7c, miR-150, miR-196b, miR-296-5p and miR-133b) that target the utrophin 3′-UTR and confirmed the previously reported targeting by miR-206 
. Importantly from a therapeutic point of view, these six miRNAs are also predicted to target utrophin in humans.
We tested whether antisense inhibition of these miRNAs could upregulate utrophin expression, and achieved this for four of the six miRNAs tested. It is not yet clear why inhibition of the other two miRNAs did not have the same effect. However, in addition to issues related to stability/chemistry of the inhibitors, some of the targeted miRNAs are only present at low levels in C2C12 cells, and therefore decreasing their expression would be predicted to have little effect on reporter construct expression.
Of the miRNAs studied, let-7c stood out as the best initial target as it is highly expressed in fast and slow skeletal muscles, and its antisense inhibition in C2C12 cells caused a 4-fold translational upregulation of the luciferase reporter. For this reason, we focused on let-7c for further experiments, and showed that blocking the binding of let-7 family miRNAs to the utrophin 3′-UTR, using a 2OMePO oligomer, could upregulate endogenous utrophin protein by over 2-fold, in C2C12 cells. The difference in the degree of response between the two experiments could be due to different time points examined (the utrophin gene is much larger than luciferase so more time was allowed to see a change in protein levels) or different amounts of transfection reagent used. Importantly, our results demonstrate that inhibition of miRNAs can de-repress the utrophin 3′-UTR and upregulate translation of utrophin protein, making it a viable therapeutic strategy for DMD.
For experiments investigating endogenous utrophin protein, we used 2OMePO oligomers designed to bind the utrophin 3′UTR and block the actions of let-7 family miRNAs, in place of commercially produced antisense miRNA inhibitors. The success of these experiments is greatly encouraging, given that 2OMePO oligomers can be synthesized on a relatively large scale are suitable for use in vivo
. Indeed, this chemistry has been used in clinical trials of exon-skipping therapeutics in patients with DMD 
. Furthermore, targeting the miRNA binding site in the utrophin 3′-UTR is likely to be more specific than targeting the miRNA itself, given that any one miRNA typically targets a number of different mRNAs.
Although our results demonstrate a crucial role of miRNAs in 3′-UTR-mediated inhibition of IRES initiation they do not exclude the possibility of other mechanisms being in operation. For example, it has recently been found that an AU-rich element in the 3′-UTR modulates utrophin mRNA stability 
In conclusion, we have shown that the utrophin-A mRNA is subject to a significant degree of translational repression, mediated by its 5′- and 3′-UTRs, and that the actions of miRNAs contribute significantly to this repression. We identify five novel miRNAs that target the utrophin 3′-UTR and demonstrate that inhibition of miRNA targeting can de-repress the utrophin 3′-UTR, leading to an upregulation in utrophin protein translation. Therefore, we believe that utrophin upregulation by miRNA inhibition represents a novel therapeutic strategy for DMD.