Search

Abstract Background Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context. Methods By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3′UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS. Results TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3′UTR-luciferase assay, confirming their interaction with the NOS1-3′UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts. Conclusion These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.