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- Title
miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context.
- Authors
Guilbaud, Marine; Gentil, Christel; Peccate, Cécile; Gargaun, Elena; Holtzmann, Isabelle; Gruszczynski, Carole; Falcone, Sestina; Mamchaoui, Kamel; Ben Yaou, Rabah; Leturcq, France; Jeanson-Leh, Laurence; Piétri-Rouxel, France
- Abstract
Background: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the <italic>DMD</italic> 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 <italic>DMD</italic> 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 <italic>NOS1</italic>, 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 <italic>NOS1</italic>-3′UTR-luciferase assay, confirming their interaction with the <italic>NOS1</italic>-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.
- Subjects
BECKER muscular dystrophy; DUCHENNE muscular dystrophy; MICRORNA; DYSTROPHIN genetics; NITRIC-oxide synthases; GENETIC mutation; GENETICS
- Publication
Skeletal Muscle, 2018, Vol 8, Issue 1, pN.PAG
- ISSN
2044-5040
- Publication type
Article
- DOI
10.1186/s13395-018-0161-2