• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of emborepLink to Publisher's site
EMBO Rep. Feb 2011; 12(2): 136–141.
Published online Jan 7, 2011. doi:  10.1038/embor.2010.208
PMCID: PMC3049433
Scientific Reports

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy

Abstract

Duchenne muscular dystrophy (DMD)—which is caused by mutations in the dystrophin gene—is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA—miR-31—that represses dystrophin expression by targeting its 3′ untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.

Keywords: DMD, dystrophin, miRNA, myoblasts, gene therapy

Introduction

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin gene. With a few exceptions, all DMD mutations (substitutions, deletions and duplications) lead to frameshifts, resulting in the formation of premature termination codons that impair dystrophin translation. When mutations do not perturb the reading frame, as in the case of Becker muscular dystrophy (BMD), the translation of a partly functional protein still occurs. As internal in-frame deletions in the protein produce only mild myopathic symptoms, a therapeutic approach based on the skipping of specific mutated exons has been conceived. This can be obtained by antisense molecules that, by pairing with splice junctions or exonic splicing enhancers, prevent exon recognition by the splicing machinery (Dunckley et al, 1998). It has been demonstrated that both chemically modified antisense oligonucleotides and antisense RNAs—derived from expression cassettes delivered through viral vectors—can rescue dystrophin synthesis in vitro (De Angelis et al, 2002; Aartsma-Rus et al, 2003) and in vivo (Bremmer-Bout et al, 2004; Goyenvalle et al, 2004; Alter et al, 2006; Denti et al, 2006, 2008). Some of these studies have now entered clinical trials (Fletcher et al, 2006; van Deutekom et al, 2007).

Recent works have shown that microRNAs (miRNAs) have a crucial role in muscle development and function (Chen et al, 2006; Naguibneva et al, 2006). Moreover, altered levels of miRNAs were found in several muscular disorders, such as myocardial infarction (Van Rooij et al, 2008), DMD and other myopathies (Eisenberg et al, 2007; Greco et al, 2009). In line with this, it has been demonstrated recently that deregulated expression of miRNAs due to a lack of dystrophin accounts for several characteristics of DMD pathogenesis (Cacchiarelli et al, 2010).

In this work we show that a specific miRNA that is highly expressed in DMD conditions represses dystrophin translation, and is therefore a potential therapeutic target for improving current strategies aimed at rescuing dystrophin synthesis and promoting fibre maturation.

Results And Discussion

Profile analysis of Duchenne compared with wild-type muscles indicated that several classes of miRNAs are differently expressed in mdx mice (Greco et al, 2009; Cacchiarelli et al, 2010). Among these, miR-31 showed a 50-fold enrichment compared with control (Fig 1A; supplementary Fig S1A online). mdx muscles are characterized by reduced fibre maturation, indicated by the decrease in miR-1 levels, and intensive regeneration, shown by the upregulation of miR-206 (supplementary Fig S1A online; Cacchiarelli et al, 2010). miR-31 and miR-206 abundance correlated with degeneration and regeneration processes, as, in muscles of 10-day-old mdx mice no upregulation was observed before the onset of dystrophic symptoms (supplementary Fig S1B online).

Figure 1
miR-31 expression. (A) miR-31 relative expression in WT and mdx mice, measured by qRT–PCR. (B) An miR-31 digoxigenin (DIG)-labelled probe was hybridized on WT and mdx gastrocnemius sections. Original magnification × 20; scale bar, 100 ...

In situ hybridization (Fig 1B) showed that miR-31 has a preferential localization in regenerating myoblasts identified by the characteristic phenotype of mononucleated fibres, which are abundant in mdx conditions and almost absent in wild type. In line with this, miR-31 localized with miR-206 (a marker of activated muscle progenitors; Cacchiarelli et al, 2010) in MyoD+/MyoG+/MHC (myosin heavy chain) cells (supplementary Fig S1C online). miR-31 was highly expressed at early stages of in vitro differentiation of wild-type mouse satellite cells, and its expression decreased progressively at later differentiation stages; by contrast, in mdx-derived satellite cells, the levels of miR-31 remained high even at prolonged differentiation times (Fig 1C). Persistent upregulation of miR-31 in mdx conditions was linked to delay of the muscle differentiation programme, as shown by longer persistence of Pax7, lower levels of MyoD and a delay in the appearance of myogenin expression in comparison with wild-type cells (supplementary Fig S2A online).

miR-31 was also more abundant in human DMD biopsies than in healthy and Becker muscles (Fig 1D). miR-31 levels remained high in DMD myoblasts induced to differentiate in vitro, whereas in healthy human controls its levels decreased with the progression of differentiation (Fig 1E). DMD myoblasts seemed to have a higher proliferating capacity and a lower differentiation potential than control cells. This was shown by the presence of PAX7 in growth conditions and the delayed appearance of differentiation markers after serum deprivation (Fig 1F). In control myoblasts, myogenin peaked at day 2 of differentiation, preceding MHC and dystrophin synthesis; in DMD cells, myogenin only appeared at day 4 preceding MHC synthesis, and was clearly visible at day 6. Immunostaining of the same samples confirmed that DMD cells express the MHC protein at lower levels and later times than wild-type cells (Fig 1G; supplementary Fig S2B online).

Altogether, these data indicate that the high levels of miR-31 in Duchenne muscles are partly due to intensive regeneration involving activated satellite cells and partly to the reduced ability of Duchenne myoblasts to complete the differentiation programme.

In silico analysis—performed by comparing messenger RNA (mRNA) expression profiles in wild-type and mdx animals (see supplementary information online)—identified many predicted targets of miR-31. Notably, the most downregulated mRNAs in mdx muscles code for proteins involved in terminal differentiation, including dystrophin (supplementary Fig S3 online). One putative binding site, embedded in a 40-nucleotide region conserved in mammalian species, was identified in the 3′ untranslated region (UTR) of the dystrophin mRNA (Fig 2A). Wild-type dystrophin 3′UTR (DMD-WT) and a derivative mutated in the miR-31 target site (DMD-mut) were fused to the luciferase open reading frame (Fig 2B). Enzyme activity was measured in C2 myoblasts in endogenous conditions and, on miR-31 overexpression, obtained through transfection of a plasmid carrying the pri-miR-31 sequence under the strong and constitutive U1snRNA promoter (pmiR-31, Fig 2C). The results indicate that miR-31 repressed luciferase activity only on DMD-WT and that de-repression was obtained with locked nucleic acid (LNA) oligos against miR-31 (LNA-31) and not with control scramble LNA. Scramble LNA transfection did not affect luciferase activities per se (not shown). Moreover, when cells were treated with a sponge construct containing four binding sites for miR-31 (Sponge-31, Fig 2A,B), luciferase activity was higher than in cells treated with a control construct (Fig 2D). Release from miR-31-repressing activity was also obtained when cells were treated with an LNA oligo complementary to 23 nucleotides across the miR-31 target site (Protector-31; Fig 2A) both in the presence of the endogenous miR-31 and in conditions of miR-31 overexpression (pmiR-31, Fig 2E). These data indicate that miR-31 targets the 3′UTR of the dystrophin mRNA and that repression is prevented either by the use of miR-31 decoys or by protecting the miR-31 binding site on dystrophin mRNA.

Figure 2
miR-31 targets dystrophin mRNA. (A) Sequences of the mature miR-31, the evolutionarily conserved 40-nucleotide region of the dystrophin 3′UTR, the decoy sequence present in the Sponge-31 construct and the 23-nucleotide-long LNA protector (Protector-31) ...

In vitro differentiation of C2 mouse myoblasts indicated that miR-31 was expressed in proliferating conditions and that its levels decreased on differentiation (Fig 3A). Synthesis of dystrophin was prominent only at day 5, although transcription was consolidated at day 3. These data indicate the expected inverse correlation between the miRNA and its target.

Figure 3
Effects of miR-31 modulation on dystrophin expression. (A) Northern (miR-31 and U2) and western (DYS and ACTN) blot analyses of C2 mouse myoblasts in growth medium (GM) and at 3 and 5 days after shift to differentiation medium. The histogram at the bottom ...

Next, we tested whether the endogenous synthesis of dystrophin responded to altered levels of miR-31 in C2 myoblasts (Fig 3B): overexpression was obtained by infection with a lentivirus containing the pri-miR-31 expression cassette (miR-31), while depletion was obtained by administration of LNA-31 or Sponge-31. When cells were induced to differentiate, a consistent decrease of dystrophin (almost threefold) was observed in conditions of persistent overexpression of miR-31; by contrast, an increase in dystrophin levels was detected when cells were treated with LNA-31 or Sponge-31. In the last two cases, the limited increase (50% and 40%, respectively) of dystrophin synthesis is probably because miR-31 levels already start to decrease at 3 days of differentiation (Fig 3A). In the lower panels of Fig 3B the levels of the dystrophin protein are compared with those of its mRNA; in all cases dystrophin mRNA levels were not affected by miR-31 modulation, indicating that the miRNA acts by repressing translation rather than controlling dystrophin mRNA stability.

DMD myoblasts, derived from a patient with deletion of exons 48–50, were infected with the U1#51 construct able to induce skipping of exon 51 and rescue of dystrophin synthesis (Fig 3C; Incitti et al, 2010). To determine whether dystrophin synthesis could be further improved by reducing miR-31 levels, we tested the ability of the Sponge-31 construct to increase dystrophin levels when infected in Δ48–50 DMD myoblasts. The sponge construct was indeed able to increase dystrophin synthesis threefold when exon skipping was applied to these cells (Fig 3D, U1#51-Sponge-31). Moreover, the anti-miR-31 LNA oligos were able to increase dystrophin synthesis twofold, when transfected into exon-skipping-treated cells (Fig 3D). Quantitative reverse transcription-polymerase chain reaction showed that dystrophin mRNA levels were the same in the different conditions, similarly to the case in muscle creatine kinase (MCK) mRNA (Fig 3D). These results indicate that dystrophin increase is mainly due to translational de-repression as a consequence of miR-31 depletion.

In conclusion, we suggest that miR-31, previously linked to neoplastic development and tumour metastasis (Valastyan & Weinberg, 2010), is part of the circuits controlling late muscle differentiation by repressing dystrophin synthesis and probably many other terminal differentiation markers. In line with this, miR-31 was previously shown to repress Myf5, a key element of muscle differentiation, in the central nervous system (Daubas et al, 2009).

miR-31-repressing activity was detected in the early phases of myoblast differentiation, supporting the idea that this control is necessary in normal muscle cells to avoid early expression of late differentiation markers and, specifically, dystrophin.

The intense and localized expression of miR-31 in regenerating myoblasts of dystrophic muscles indicates that the high levels of miR-31 found in dystrophic conditions—in both mouse and human biopsies—are due to the intensive regeneration programme that is mediated by the activation of satellite cells. Interestingly, in dystrophic myoblasts and satellite cells the lack of dystrophin correlates with a delay of the maturation process of the cells.

In this study we have also shown that in dystrophic conditions, when dystrophin synthesis is rescued through exon skipping, the inhibition of miR-31 activity increased dystrophin production. This might reflect both enhanced differentiation as well as enhanced translation of the dystrophin mRNA.

The contribution to dystrophin production by regenerating fibres in a compromised muscle is important. miR-31 repression in this compartment could therefore improves therapeutic treatments aimed at increasing levels of dystrophin synthesis. Rescue of consistent levels of dystrophin will have additional benefits, such as completion of the muscle fibre maturation process.

Methods

RNA and protein analyses. Total RNA was prepared from liquid nitrogen-powdered tissues homogenized in Qiazol reagent (QIAGEN). miRNA and mRNA analyses were performed using miScript System (QIAGEN). Relative quantification was performed using, as endogenous controls, U6 snRNA for miRNAs and HPRT1 for mRNAs. Northern blots for miRNAs were performed using LNA detection probes (Exiqon). miRNA in situ hybridization was performed in formaldehyde and carbodiimide (EDC)-fixed gastrocnemius cryosections, according to Pena et al (2009). Western blot on total extracts and immunohistochemistries were performed as described in Denti et al (2006).

Luciferase constructs and assays. Full-length murine DMD-3′UTR sequence (DMD-WT, 2,461 bp) was amplified by polymerase chain reaction and then cloned in NotI restriction site of the psicheck2 plasmid (Promega), downstream from the renilla luciferase (RLuc) gene. The same plasmid also contains the firefly luciferase (FLuc) to normalize transfection efficiency. Mutant derivative DMD-mut was obtained by deletion of miR-31 binding sites. RLuc and FLuc activities were measured by Dual Glo luciferase assay (Promega).

miRNA overexpression and sponge constructs. Plasmid pmiR-31 was produced by cloning a pri-miR-31 with 100 nucleotides upstream and downstream from the pre-miRNA into the U1snRNA expression cassette (Denti et al, 2004). Sponge-31 construct was generated by cloning-annealed oligonucleotides containing four artificial miR-31 binding sites (sequence in Fig 2A) into the WPRE SacII restriction site downstream from the green fluorescent protein open reading frame, according to Gentner et al (2009). miR-31 sponge was combined with exon skipping by cloning-annealed oligos into the WPRE sequence of the green fluorescent protein reporter of lentiviral PCCL-U1#51 reported to induce skipping of DMD exon 51 (Incitti et al, 2010). Oligonucleotide sequences and plasmids are available on request.

Cell culture. Muscle satellite cells were cultured and differentiated as described in Rando & Blau (1994). The Duchenne primary myoblasts carrying deletion of exons 48–50 were obtained from Telethon Biobank and infected with a lentiviral construct containing antisense sequences against the splice junctions of exon 51. LNA miRNA knockdown (Exiqon) treatments were performed according to Taulli et al (2009).

Statistical analyses. The data shown in the histograms are the result of at least three independent experiments performed on at least three samples or animals. Data are shown as mean±standard deviation. Unless stated otherwise, the statistical significance of differences between means was assessed by two-tailed t-test and P<0.05 was considered significant.

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

Supplementary Information:

Acknowledgments

We thank Professor A. Musarò, Dr E. Bertini, Dr A. D'Amico, Dr M. Mora and the Telethon Neuromuscular Biobank for providing material. We also thank M. Marchioni for technical support. D.C. is a recipient of a Microsoft research PhD fellowship. This work was partly supported by grants from Telethon (GGP07049), Parent Project Italia, EU project SIROCCO (LSHG-CT-2006-037900), ESF project ‘NuRNASu', IIT ‘SEED', PRIN and BEMM.

Footnotes

The authors declare that they have no conflict of interest.

References

  • Aartsma-Rus A, Janson AA, Kaman WE, Bremmer-Bout M, den Dunnen JT, Baas F, van Ommen GJ, van Deutekom JC (2003) Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 12: 907–914 [PubMed]
  • Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD, Partridge TA, Lu QL (2006) Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med 12: 175–177 [PubMed]
  • Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ, Kaman WE, Janson AA, Vossen RH, van Ommen GJ, den Dunnen JT, van Deutekom JC (2004) Targeted exon skipping in transgenic hDMD mice: a model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther 10: 232–240 [PubMed]
  • Cacchiarelli D et al. (2010) microRNAs involved in molecular circuitries relevant for the Duchenne muscular dystrophy pathogenesis are controlled by the dystrophin/nNOS pathway. Cell Metab 12: 341–351 [PubMed]
  • Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, Conlon FL, Wang DZ (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 38: 228–233 [PMC free article] [PubMed]
  • Daubas P, Crist CG, Bajard L, Relaix F, Pecnard E, Rocancourt D, Buckingham M (2009) The regulatory mechanisms that underlie inappropriate transcription of the myogenic determination gene Myf5 in the central nervous system. Dev Biol 327: 71–82 [PubMed]
  • De Angelis FG, Sthandier O, Berarducci B, Toso S, Galluzzi G, Ricci E, Cossu G, Bozzoni I (2002) Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a corrected phenotype in Δ48–50 DMD cells. Proc Natl Acad Sci USA 99: 9456–9461 [PMC free article] [PubMed]
  • Denti MA, Rosa A, Sthandier O, De Angelis FG, Bozzoni I (2004) A novel vector for siRNA expression in mammalian cells that relies on polII-dependent transcription. Mol Ther 10: 191–199 [PubMed]
  • Denti MA et al. (2006) Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model. Proc Natl Acad Sci USA 103: 3758–3763 [PMC free article] [PubMed]
  • Denti MA, Incitti T, Sthandier O, Nicoletti C, De Angelis FG, Rizzuto E, Auricchio A, Musarò A, Bozzoni I (2008) Long-term benefit of adeno-associated virus/antisense-mediated exon skipping in dystrophic mice. Hum Gene Ther 19: 601–608 [PubMed]
  • Dunckley MG, Manoharan M, Villiet P, Eperon IC, Dickson G (1998) Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet 7: 1083–1090 [PubMed]
  • Eisenberg I et al. (2007) Distinctive patterns of microRNA expression in primary muscular disorders. Proc Natl Acad Sci USA 104: 17016–17021 [PMC free article] [PubMed]
  • Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Wilton SD (2006) Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide. J Gene Med 8: 207–216 [PubMed]
  • Gentner B, Schira G, Giustacchini A, Amendola M, Brown BD, Ponzoni M, Naldini L (2009) Stable knockdown of microRNA in vivo by lentiviral vectors. Nat Methods 6: 63–66 [PubMed]
  • Goyenvalle A, Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L, Danos O (2004) Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306: 1796–1799 [PubMed]
  • Greco S et al. (2009) Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia. FASEB J 23: 3335–3346 [PubMed]
  • Incitti T, De Angelis FG, Cazzella V, Sthandier O, Pinnarò C, Legnini I, Bozzoni I (2010) Exon skipping and Duchenne muscular dystrophy therapy: selection of the most active U1 snRNA-antisense able to induce dystrophin exon 51 skipping. Mol Ther 18: 1675–1682 [PMC free article] [PubMed]
  • Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, Souidi M, Cuvellier S, Harel-Bellan A (2006) The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat Cell Biol 8: 278–284 [PubMed]
  • Pena JT et al. (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6: 139–141 [PMC free article] [PubMed]
  • Rando TA, Blau HM (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125: 1275–1287 [PMC free article] [PubMed]
  • Taulli R, Bersani F, Foglizzo V, Linari A, Vigna E, Ladanyi M, Tuschl T, Ponzetto C (2009) The muscle-specific microRNA miR-206 blocks human rhabdomyosarcoma growth in xenotransplanted mice by promoting myogenic differentiation. J Clin Invest 119: 2366–2378 [PMC free article] [PubMed]
  • Valastyan S, Weinberg RA (2010) miR-31: a crucial overseer of tumor metastasis and other emerging roles. Cell Cycle 9: 2124–2129 [PubMed]
  • van Deutekom JC et al. (2007) Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med 357: 2677–2686 [PubMed]
  • Van Rooij E, Marshall WS, Olson EN (2008) Toward microRNA-based therapeutics for heart disease: the sense in antisense. Circ Res 103: 919–928 [PMC free article] [PubMed]

Articles from EMBO Reports are provided here courtesy of The European Molecular Biology Organization
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...