miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context
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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-04-01
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| Series: | Skeletal Muscle |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13395-018-0161-2 |
| _version_ | 1798045929478029312 |
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| author | Marine Guilbaud Christel Gentil Cécile Peccate Elena Gargaun Isabelle Holtzmann Carole Gruszczynski Sestina Falcone Kamel Mamchaoui Rabah Ben Yaou France Leturcq Laurence Jeanson-Leh France Piétri-Rouxel |
| author_facet | Marine Guilbaud Christel Gentil Cécile Peccate Elena Gargaun Isabelle Holtzmann Carole Gruszczynski Sestina Falcone Kamel Mamchaoui Rabah Ben Yaou France Leturcq Laurence Jeanson-Leh France Piétri-Rouxel |
| author_sort | Marine Guilbaud |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T23:30:04Z |
| format | Article |
| id | doaj.art-8dc62b062dec4a498160c96171b1ce67 |
| institution | Directory Open Access Journal |
| issn | 2044-5040 |
| language | English |
| last_indexed | 2024-04-11T23:30:04Z |
| publishDate | 2018-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Skeletal Muscle |
| spelling | doaj.art-8dc62b062dec4a498160c96171b1ce672022-12-22T03:57:11ZengBMCSkeletal Muscle2044-50402018-04-018111310.1186/s13395-018-0161-2miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic contextMarine Guilbaud0Christel Gentil1Cécile Peccate2Elena Gargaun3Isabelle Holtzmann4Carole Gruszczynski5Sestina Falcone6Kamel Mamchaoui7Rabah Ben Yaou8France Leturcq9Laurence Jeanson-Leh10France Piétri-Rouxel11Sorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieSorbonne Université-UMRS974-Inserm-Institut de MyologieLaboratoire de Génétique et Biologie Moléculaire, Hôpital CochinGénéthonSorbonne Université-UMRS974-Inserm-Institut de MyologieAbstract 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.http://link.springer.com/article/10.1186/s13395-018-0161-2Duchenne muscular dystrophy (DMD)Becker muscular dystrophy (BMD)miRNAnNOS |
| spellingShingle | Marine Guilbaud Christel Gentil Cécile Peccate Elena Gargaun Isabelle Holtzmann Carole Gruszczynski Sestina Falcone Kamel Mamchaoui Rabah Ben Yaou France Leturcq Laurence Jeanson-Leh France Piétri-Rouxel miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context Skeletal Muscle Duchenne muscular dystrophy (DMD) Becker muscular dystrophy (BMD) miRNA nNOS |
| title | miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context |
| title_full | miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context |
| title_fullStr | miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context |
| title_full_unstemmed | miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context |
| title_short | miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context |
| title_sort | mir 708 5p and mir 34c 5p are involved in nnos regulation in dystrophic context |
| topic | Duchenne muscular dystrophy (DMD) Becker muscular dystrophy (BMD) miRNA nNOS |
| url | http://link.springer.com/article/10.1186/s13395-018-0161-2 |
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