miR-206 is a member of the muscle-specific miR-1 family, that consists of six members clustered into three bicistronic pairs arising from an initial local gene duplication which produced the original paralogous gene cluster (miR-1 and miR-133). Then two "non-local" genomic duplications resulted in the new clusters located on different chromosomes . It is the unique myomiR exclusively expressed in skeletal muscle [32–36] and has been rarely detectable in the heart [37–41]. The skeletal muscle-specific expression of miR-206 was first clearly demonstrated by microarray analysis and later confirmed by Northern blot .
Additional muscle-enriched miRNAs have also been identified and shown to be involved in cardiogenesis, myogenic, differentiation and growth . Several studies were performed to analyze the expression of miRNAs, in general, and myo-miRs, specifically, in muscolar dystrophies. Eisenberg et al.  performed a microarray analysis on 10 muscular disorders in humans, not including DM1. They identified 185 miRNAs with differential expression, but myomiRs were not included in this list. Microarray analyses of muscle from the dystrophin-deficient (mdx) mouse, an animal model of Duchenne muscular dystrophy (DMD), suggest that changes in miRNAs expression may contribute to the pathophysiology of muscular dystrophy [42–45]. Therefore, McCarthy et al.  analyzed the expression of the muscle-enriched miRNAs in the mdx diaphragm, the most severely affected muscle in the dystrophin-deficient mouse. They observed an increase in miR-206 expression in this muscle, associated with a similar increase in Myod1 expression. These results suggested that miR-206 expression contributes to the chronic pathology observed in the mdx diaphragm by repressing expression of genes that otherwise would serve a compensatory function, limiting the severity of the disease, as in the hindlimb musculature .
The main goal of this paper was to investigate the pathophysiological roles of muscle-specific miRNAs in DM1, the most frequent autosomal dominant myopathy in adults. We therefore profiled the expression of miR-133, miR-1, miR-181 and miR-206, in 7 vastus lateralis biopsies from DM1 patients compared with 4 control subjects. We have also included in our study the muscular expression of the miR-103 and miR-107 CTG-repeat binding miRNAs which are highly expressed in brain, heart and muscle . These two miRNAs contain CAG repeats in their seed regions and have been identified, through computational analysis, as potential repressor factors of the wild type and mutant DMPK transcripts. The binding of miR-103 and miR-107 to the 3'UTR of the DMPK expanded mRNAs could therefore affects the stoichiometry of free to bound CTG-repeat binding miRNAs, or otherwise disrupt the CTG-repeat binding miRNA function in DM1 muscle tissues.
After a combination of QRT-PCR and Northern blot experiments, only miR-206 was found to be over-expresssed in 5 of 7 DM1 patients compared with the controls group. Interestingly, samples DM1-5 and DM1-7, which did not show upregulation of miR-206, demonstrated lower phosphatase activity and milder atrophy compared to the other DM1 specimens. The misregulation of miR-206 in DM1 is consistent with what observed by McCarthy et al.  in the affected diaphragm of mdx mouse. Since the vastus lateralis from DM1 patients exhibits all the pathological hallmarks of a dystrophic tissue, miR-206 may contribute to the chronic course of both muscular dystrophies. Several computational and functional studies identified the putative targets of miR-206. Rosenberg et al.  have predicted its targets based on sequence match, and indicated the p180 subunit of DNA polymerase α and three other genes as direct targets. Down-regulation of the polymerase inhibits DNA synthesis, an important component of the differentiation program, connecting miR-206 function to the cell quiescence in the differentiation process. Moreover they showed that miR-206 was capable of post-transcriptionally repressing Utrophin expression. They concluded that these data could be used to develop specific therapies aimed at increasing or maintaining Utrn expression in Duchenne muscular dystrophy.
To determine whether miR-206 might function in a similar fashion under dystrophic conditions, John J. McCarthy et al. measured Utrophin protein levels in mdx diaphragm . In this study the Utrophin transcript level has also been evaluated, since there is increasing evidence that miRNAs can also accelerate target mRNA degradation  with the consequent decreasing of target mRNA abundance. Results indicate that Utrophin is post-transcriptionally regulated in the mdx diaphragm, but are not consistent with regulation by miR-206 as Utrophin protein increased, not decreased as would be expected if regulated by miR-206. Similarly, we tested the protein and mRNA levels of Utrophin in muscle biopsies from DM1 patients showing a miR-206 over-expression. Western blot and QRT-PCR analyses did not demonstrate significant differences between DM1 and controls groups. Our observation further support the idea that the Utrophin gene is not target of miR-206 in vivo in our DM1 muscle samples.
Hypothetical mRNA targets of miR-206 can also be derived, trough computational analysis, from microarray studies of mRNA differentially expressed in DM1 tissues. Osborne at al.  performed a global mRNA profiling in transgenic mice that express CUGexp RNA to identify DM1-affected genes and study mechanisms for dysregulation. 175 transcripts were dysregulated in this mice models, comprising 110 transcripts that were upregulated and 65 that were downregulated. In-silico analysis through Targetscan http://www.targetscan.org indicate that five of the downregulated transcripts are potential target of miR-206: RETSAT (all trans retinol 13,14 reductase), GNPNAT1 (glucosamine-phosphate N-acetyltransferase 1), LAPTM4B (lysosomal-associated protein transmembrane 4B), IGFBP5 (insulin-like growth factor binding protein 5) and VASP (vasodilator-stimulated phosphoprotein) mRNAs. It is therefore possible that the effect of miR-206 upregulation found in our DM1 sample could influence the expression of additional genes not reported so far in literature.
Even if data about the target of miRNAs are increasing, less is know about the distribution and localization of miRNAs in the cells. Politz et al. described the intracellular localization of miR-206 in single cultured myogenic cells using in situ hybridization followed by high-resolution imaging microscopy. They found that miR-206 is not only distributed throughout the cytoplasm as expected but also is concentrated in the nucleolus .
To detect and localize miR-206 in our DM1 and control muscle biopsies, we exploited the higher specificity and hybridization efficiency of locked nucleic acid (LNA) probes. These LNA-modified molecules exhibit unprecedented thermal stability when hybridized with their RNA target molecules. The analysis of miRNAs accumulation in frozen tissue sections using (DIG)-labeled LNA probes resulted in the generation of comprehensive miRNA expression atlases that have proven highly useful for functional studies of individual miRNA .
We therefore utilized the same technology to determine the tissue localization of miR-206 in transversal section of vastus lateralis from DM1 and control subjects. Interestingly, we found that miR-206 is prevalently expressed in the nuclear regions, with a tissue distribution in DM1 muscles characterized by a strong signal corresponding also to the nuclear clumps and centralized nuclei. The localization of miR-206 in DM1 atrophic fibers may indicate a possible involvement of miR-206 in the process of atrophy which already involves the activation of the MyomiRs network in the regulation of slow myosin expression .
Also if deeper studies need to be performed in order to improve our knowledge on miR-206 involvement in DM1, it is possible to speculate that miR-206 could contribute to the chronic course of the pathology and need to be considered for future molecular therapies.