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. Keywords: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), miRNA, nNOS * Correspondence: email@example.com Sorbonne Université-UMRS974-Inserm-Institut de Myologie, 105 bd de l’Hôpital, 75013 Paris, France Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 2 of 13 Background location was described to contribute to DMD pathophysi- Duchenne muscular dystrophy (DMD) is a progressive and ology by the disturbance of NO signaling leading to an in- fatal X-linked neuromuscular disorder caused by mutations crease of the muscle fatigability [20, 21]. in dystrophin gene (DMD)[1, 2]. The disease is due to mu- Our goal was to identify the molecular factors that can tations that disrupt the translational reading frame, leading modulate the expression of nNOS and the muscular bi- to the loss of the protein dystrophin expression. Mutations opsies of BMDd45-55 patients that were sought to be a in the DMD gene that preserve the open reading frame pertinent tool. Indeed, in these patients, the deletion of allow the production of an abnormal truncated dystrophin the 45–55 exons in dystrophin mRNA should partially still retaining some functional capacity, leading to a milder delete the spectrin motif repeat 17 in the resulting pro- muscle disease (Becker muscular dystrophy or BMD) [3, 4]. tein and could alter the nNOSμ anchoring. A previous This feature is the rationale of exon skipping therapy and study revealed that the BMDd45-55 patients displayed genome editing strategies now in development [5–7]. The variable clinical and histological phenotypes and that a principle of these approaches is to delete one or multiple subsequent decrease of nNOS protein expression oc- exons in order to obtain the production of a truncated dys- curred in these patients compared to healthy subjects trophin, inducing a phenotypic conversion of DMD into . Furthermore, several studies demonstrated a de- BMD. To apply these strategies to a larger number of eli- regulation of miRNA expression profiles in dystrophino- giblepatients, theskipping of exons45to55ofthe DMD pathies [14, 23–26]. Cacchiarelli et al.’s study showed gene has been proposed since that could correct the read- also that the loss of nNOS sarcolemmal localization ing frame in about 63% of DMD patients with deletions [8, leads to the deregulation of the expression of several 9]. Given the perspective of this approach, the therapeutic microRNAs (miRNAs) . miRNAs are short noncod- relevance of the resulting 45–55 truncated dystrophin may ing RNA that regulate mRNA post-transcriptionally ei- be deduced from the clinical status of BMD patients carry- ther by promoting mRNA degradation or by inhibiting ing spontaneous deletion of exons 45 to 55 of the DMD protein translation . miRNAs have been shown to gene (BMDd45-55). Likewise, molecular investigations of regulate functions of the skeletal muscle both in normal factors involved in pathophysiological process occurring in and pathological states [14, 28–30]. Altogether, these muscle of these patients are of great interest. studies suggest a link between miRNA expression, nNOS Dystrophin is a 427-kDa protein that links the cytoskel- expression, and physiopathology of dystrophinopathies. eton to sarcolemma via the dystrophin-associated protein Thus, the aim of the present study was to identify miR- complex (DAPC) . DAPC provides stability and integ- NAs that could modulate nNOS expression by screening rity to the muscle membrane during contraction. The loss the miRNA profile in BMDd45-55 muscular biopsies. of dystrophin leads to a breakdown of the DAPC complex, and as consequences, the muscle fibers become more sen- Methods sitive to mechanical stresses, leading to muscle degener- Ethics approvals ation, chronic inflammation, or increased fibrosis [11, 12]. Muscle biopsies were collected from patients after in- Among the partners of the dystrophin, the neuronal nitric formed consent, and this study was performed with oxide synthase (nNOS), that synthesizes nitric oxide (NO) agreement from the Committee for the Protection of , was shown to play critical roles in a variety of muscle Persons (CPP) concerned. functions, including not only contraction, regeneration, at- rophy, glucose uptake, and blood perfusion  but also Cohort of patients transcriptional regulation . Indeed, NOS enzymatic ac- Nine Becker muscular dystrophy (BMD) patients charac- tivity was recently demonstrated as essential for the rescue terized for a deletion of exons 45–55 of the DMD gene of muscle mass after atrophy induced by unloading , were studied. These patients were already described . as well as in reducing the extent of atrophy during disease Indeed, the clinical status of the patients was scored , and these effects were mostly assigned to activation using the Gardner–Medwin and Walton scale (GMWS) of muscle stem cells by the NO production. Three differ- , and the histopathological status based on routine ent isoforms of nNOS, namely nNOSα,nNOSβ,and hematoxylin and eosin (HE) staining of muscle cryosec- nNOSμ, were described to be expressed in the skeletal tions has been investigated showing a large histological muscle. The nNOSμ, the major one, contains a PDZ do- disparity. These criteria allowed defining three classes of main which allows its binding to the rod domain of the severity: (i) “mild” (GMWS ≤ 2 [i.e., still able to normally dystrophin at the spectrin-like repeats 16 and 17 (R16/17) climb stairs] and normal muscle biopsy); (ii) “moderate” encoded by exons 42–45 . It has been shown that in (GMWS ≤ 2 or mild dystrophic muscle biopsy); (iii) “se- the absence of dystrophin, nNOSμ was delocalized from vere” + (GMWS ≤ 2 and/or dystrophic muscle biopsy) the sarcolemma of the muscular fibers and its expression (Table 1). The 9 patients were biopsied at an age ranging decreased [18, 19]. Alteration of nNOSμ expression and from 9 to 69 years old for diagnostic purposes after Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 3 of 13 Table 1 Clinical and histopathological phenotypes compared with nNOSμ expression in BMDd45-55 patients Name Severity class Age at muscle biopsy Histopathological status (*) nNOSμ protein expression (**) Ctrl 1 26 N ++ Ctrl 2 40 N ++ Ctrl 3 10 N ++ Ctrl 4 N ++ Ctrl 5 N ++ Patients BMD 1 Moderate 35 +/− (35) +/− BMD 2 Severe + 13 + (13) + BMD 4 Severe + 33 ++ (33) + BMD 7 Moderate 40 +/− (40) + BMD 8 Moderate 12 +/− (12) +/− BMD 11 Mild 69 N (69) + BMD 12 Moderate 18 +/− (18) +/− BMD 18 ND BMD 31 ND (*) N normal, +/− mild dystrophy, + and ++ severe dystrophy, ND not determined, (**) +/− traces or not detectable in Western blot, + weak, ++ normal informed consent, and these biopsies were used during to the manufacturer’s instructions. Complementary DNAs the experiments in the present article. In addition, the (cDNAs) were analyzed by real-time quantitative PCR per- five muscle biopsies used as healthy control muscles formed on Light Cycler 480 instrument (Roche) using Exi- (Ctrl) were recovered as surgical wastes from orthopedic lent SybR Green Master Mix (Exiqon). LNA™ PCR surgery of individuals without neuromuscular diseases. Primers set from Exiqon were used for miRNA expression All human muscle biopsies were flash frozen in isopen- analysis (miR-212: 204170, miR-708: 204490, miR-34c: tane cooled in liquid nitrogen and evaluated for dys- 205659, miR-31: 204236). miRNA expression was normal- −dCT trophin and nNOS expression by Western blotting . ized on miR-30b-5p expression (204765) using 2 method. Taqman Low-Density Array (TLDA) Total RNA (including miRNA and mRNA) were ex- Luciferase assay tracted from about 30 mg of muscular biopsy using the Genomic DNA was extracted from biopsies of healthy NucleoSpin© miRNA kit from Macherey-Nagel. Total subject using NucleoSpin© Tissue kit from Macherey- RNA (200 ng) was reverse-transcribed with the Mega- Nagel, following the manufacturer’s instructions. gDNA plex Primer Pools A and B (human version 3), and miR- was eluted after incubation of the silica membrane 3 min, NAs were quantified after a pre-amplification step, with twice in 50 μL of Elution Buffer BE, followed by centrifu- TaqMan Array MicroRNA Cards A and B (human ver- gation 1 min at 11,000g, being 100 μl of total gDNA. sion 3) on the 7900HT Real-Time PCR System (AB) ac- NOS1-3′UTR (Ensembl: ENST00000618760) was cording to the manufacturer’s guidelines. Relative cloned downstream of Firefly luciferase gene in HSVTK- −dCT quantification was performed with 2 method, using Luc3′ modified plasmid. As this 3′UTR is 7183 pb in the mean of all miRNA expressed as normalizer. length, it is too large to be fully cloned in this plasmid. Therefore, we cut it in 4 overlapping parts using follow- miR-target predictions ing primers that add restriction sites (in bold) on gDNA The ability of candidate miRNAs to target NOS1-3′UTR was fragments (Table 2). evaluated with Diana-microT algorithm and TargetScan v6.2. Fragments were amplified using Mastermix Phusion with the following protocol: 98 °C 30s; 10 cycles of 98 °C Individual RT-qPCR 10s, 58 °C 30s, and 72 °C 1 min; and 20 cycles of 98 °C Thirty milligrams of muscular biopsies from 3 healthy 10s, 61 °C 30s, 72 °C 1 min, and 72 °C 10 min. subjects and patients BMD1, 2, 4, 8, and 11 was extracted Amplicons were purified with NucleoSpin© Gel and using the NucleoSpin© miRNA kit (Macherey-Nagel). PCR cleanup from Macherey-Nagel. 3′UTR parts were One hundred nanograms of RNA was reverse-transcribed then cloned in HSVTK-Luc3′ modified plasmid between with Universal cDNA synthesis kit II (Exiqon) according XbaI and EcorV sites. Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 4 of 13 Table 2 Primers used to fragment NOS1-3′UTR Position in 3′UTR) Forward primer Reverse primer Part 1 (1-1896) F1-5′TTGTCTAGACTGGACCCTCTTGCCCAGC-3′ R1-5′AAGGATATCCAGGGGAAATTGGGATTAAAGG-3′ Part 2 (1773-3685) F2-5′-AACTCTAGACTATGACTCACCTTGCTCTGC-3′ R2-5′-ATCGATATCCTTACATGCTCCCTGTCCGTG-3′ Part 3 (3607-5523) F3-5′-AATTCTAGACTGGTAGCTTCTGGAAGGTAAG-3′ R3-5′AATGATATCGCCACAAGGCAGGGACTGGC-3′ Part 4 (5358-7149) F4-5′-TAGTCTAGAGAAACACAGGTCTGAGGGTCTG-3′ R4-5′-CCGATATCATTGTAACCATAATGCAAACAAGC-3′ Added restriction sites are indicated in bold characters (XbaI and EcorV) Each 3′UTR construction (24.5 ng) was co-transfected miR-31: 204236). miRNA expression was normalized on −dCT in 293T-HEK cells with 25 pg of either miR-negative SNORD44 expression (203902) using 2 method. control (AM17111, Ambion) or miR-212, miR-31, miR- 34c, or miR-708 (AM17100, Ambion) using lipofecta- Western blotting mine 2000 diluted in Optimem reduced medium. The For nNOS detection, cells were lysed in 50 μl of RIPA plasmid CMV-Renilla luciferase (0.25 ng) was also trans- buffer (150 mM NaCl,50 mM 4-(2-hydroxyethyl)-1- fected in each condition as normalizer. Five hours post- piperazineethanesulfonic acid, pH 7.4, 5 mM ethylene transfection, Optimem reduced medium is replaced with diamine tetra acetic acid, 1% NP-40, 0.5% sodium deoxy- DMEM added with FBS 10%. Twenty-four hours after cholate, 0.1% sodium dodecyl sulfate, 1 mM PMSF with transfection Firefly and Renilla luciferase luminescences a mix of protease inhibitors (Roche), and centrifuged were quantified with Dual-Glo Luciferase Assay System 10 min 1500g at 4 °C. (according to the manufacturer’s instructions) on Flex- Protein extracts (20 μg) were denaturated in Laemmli station 3 Microplate reader. Firefly luciferase activity buffer 2× added of 10% of 2-mercaptoethanol 30 min at was normalized on Renilla luciferase activity. room temperature (RT) and incubated 15 min in ice and then 15 min at RT. Proteins were resolved by SDS– Human myoblast transfection PAGE (4–12%, Invitrogen) and transferred to nitrocellu- Human immortalized myoblasts from a healthy subject lose. Membranes were blocked in tris-buffered saline 0. (ctrl) and from a DMDd45-52 patient (DMDd45-52) were 1% Tween-20 with 5% non-fat dry milk 1 h at RT and in- used . Myoblasts were plated 48 h before transfection cubated overnight at 4 °C, with rabbit polyclonal nNOS 4 3 at 3 × 10 /well of 6-well plate or 6 × 10 /well of 24-well antibody (R-20, Santa Cruz, 1:100) or with mouse mono- plate in proliferation medium composed of DMEM supple- clonal GAPDH antibody (MAB9748, Tebu-Bio, 1:8000). mented with 5 μg/ml of insulin, 5 ng/ml of EGF, 0.5 ng/ml After being washed in TBS 0.1% Tween, membranes of bFGF, 0.2 μg/ml of dexamethasone, 25 μg/ml of fetuin, were incubated for 1 h at RT with secondary antibodies: 20% of fetal bovine serum, and 16% of medium 199. Cells goat anti-rabbit-horseradish peroxidase (HRP) (1/50000) were transfected with 12.5 pg of either miR-negative con- or sheep anti-mouse HRP (1/15000) (Jackson Immunor- trol (AM17111, Ambion); miR-31, miR-34c, or miR-708 esearch). Western blots were revealed with enhanced (AM17100, Ambion); or antimiR-34c or antimiR-708 chemiluminescence (Thermo Scientific) with Image (AM17000, Ambion) using lipofectamine 2000 diluted in Quant LAS 4000 system (GE Healthcare Life Sciences). Optimem reduced medium. Twenty-four hours after trans- fection, transfection medium was replaced with prolifera- Immunostaining tion medium for 24 h. Cells in 24-well plate were washed with PBS and fixed with paraformaldehyde 4% 10 min at RT and washed 3 miRNA expression times in PBS. Fixed cells were permeabilized with 0.5% Cells were harvested in 300 μl of Buffer ML (NucleoSpin© Triton X-100 (Sigma-Aldrich), washed, and blocked in miRNA, Macherey-Nagel). Total RNA (small + large PBS/5% bovine serum albumin (BSA) for 40 min at RT. RNA) was extracted from lysed cells with NucleoSpin© Cells were then incubated in PBS/1% BSA/0.1% saponin miRNA kit (Macherey-Nagel) following the manufac- with a goat polyclonal anti-nNOS antibody (Ab1376, turer’s instructions. cDNA generated with Universal Abcam, 1:500), overnight at RT; washed in PBS/1%BSA/ cDNA synthesis kit II (Exiqon) according to the manufac- 0.1% saponin; and incubated for 1 h with secondary anti- turer’s instructions was analyzed by real-time quantitative body: Donkey anti-goat (Alexafluor 594 conjugate, Life PCR performed on Light Cycler® 480 instrument (Roche) Technologies, 1:500) and with DAPI (1:5000, Sigma). using Exilent SybR Green Master Mix (Exiqon). LNA™ Fixed cells were then thoroughly washed in PBS/1% PCR Primers set from Exiqon were used for miRNA ex- BSA/0.1% saponin and then in PBS and mounted in pression analysis (miR-708: 204490, miR-34c: 205659, Fluoromount (Southern Biotech). Images were acquired Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 5 of 13 with Leica DM2500 confocal microscope using × 63 miRNAs using Taqman Low-Density Array (TLDA) in objective. muscle biopsies of 9 BMDd45-55 patients, compared to 5 control subjects (Table 1). From TLDA data Statistical analysis (Additional file 2), we established a list of miRNAs Statistical analysis were performed using Student’s t test. overexpressed in the muscles of BMDd45-55 patients A value of p < 0.05 was considered statistically significant. with the criteria of a fold change higher than 2 and a Methods used for the Additional file 4: Figure S1 are p value less than or equal to 0.05. By comparing described in Additional file 1. miRNA expression levels between BMDd45-55 and healthy muscles as control (Fig. 1a), a total of 66 miR- Results NAs were identified based on the defined criteria of miRNA expression profiling in BMDd45-55 muscular fold change and p value. Furthermore, the TLDA data biopsies were also analyzed by comparing the level of miRNAs To start our analysis, we took advantage of having a expressed in muscles of severe patients with those collection of muscle biopsies of Becker patients expressed in muscles of all the other patients (Fig. 1b). (BMDd45-55) bearing an in-frame deletion of exons 45 This analysis allowed the identification of 29 overex- to 55 in the DMD gene and well characterized from a pressed miRNAs. It should be noted that none of these genetic point of view . This collection has been the 29 miRNAs were found in the list of miRNAs overex- subject of a preliminary study which showed notably a pressed in BMD muscles compared to healthy subjects decrease in the expression of the protein nNOS in the (Fig. 1a), probably because of the too small number of muscle of the studied patients . We examine here a severe muscle biopsies preventing the fold change value potential role of miRNA in the regulation of nNOS ex- from being statistically significant when included to the pression by investigating the expression levels of 617 values obtained for all the BMD patients. Fig. 1 Screening of miRNA expression profiling by TLDA in BMDd45-55 muscular biopsies. Data of TLDA were expressed by the value of Log2(R), where R is the ratio of the average of the relative quantification (RQ) obtained in BMDd45-55 muscles on the average of RQ values obtained from muscle of healthy subjects (a), or obtained from muscle of severe BMDd45-55 patients on the average of RQ values obtained from muscle of the moderate and mild BMDd45-55 patients (b), p ≤ 0.05. RQ are obtained using average of values of all miRNA for normalizer Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 6 of 13 miR-708-5p, miR-31-5p, and miR-34c-5p target 3′UTR patients compared to control muscle or in severe pa- sequences of NOS1 gene tients compared to other patients and that only 3 of To select miRNAs that could modulate nNOS expres- them could target sequences present in the NOS1-3′ sion, the total sequence of the 3′UTR of the NOS1 gene UTR and modulate reporter gene activity. (NOS1-3′UTR) was submitted to two predictive soft- ware, i.e., TargetScan Human and microRNA.org, that miR-31, miR-708, and miR-34c effect on nNOS expression process alignment of the target sequence with human in human myoblasts miRNA databases (Fig. 2a). From this study, 12 and 24 In order to address the causal relationship between the miRNAs were identified by the 2 predictive software, re- overexpression of the 3 selected miRNAs and the nNOS spectively. Surprisingly, there was no common miRNAs expression in muscular context, we carried out experi- between the two lists. By combining the previous TLDA ments using immortalized human myoblasts from healthy analysis criteria and the in silico investigation data, 4 subject (control) and from a patient displaying a deletion miRNAs named miR-31-5p (miR-31), miR-708-5p (miR- of the exons 45 to 52 (DMDd45-52) in the DMD gene 708), miR-34c-5p (miR-34c), and miR-212-3p (miR-212) . First, these DMDd45-52 myoblasts were validated as were finally selected. Overexpression of these 4 miRNAs an appropriate cellular model regarding the expression of was then validated by performing individual RT-qPCR the 3 selected miRNAs. Quantification by RT-qPCR con- on 5 BMDd45-55 and 3 healthy muscular biopsies (Fig. firmed a higher level of expression for miR-31, miR-708, 2c). A higher level of expression of the 4 miRNAs was and miR-34c in DMDd45-52 cells compared to control detected in BMDd45-55 compared to control muscles with a fold change of 2.2, 2.2, and 3.8, respectively (Fig. with a fold change of 6.6, 4.4, 10.1, and 3.3 for miR-31, 4a). Furthermore, the expression of nNOS protein was in- miR-708, miR-34c, and miR-212, respectively, confirm- vestigated by Western blot and showed a significant de- ing the results obtained by TLDA (Fig. 2b, crease in DMDd45-52 compared to control cells (Fig. 4b). Additional file 2). Furthermore, by analyzing the se- Additionally, immunostaining experiments, allowing the quence of the NOS1-3′UTR regarding the 4 selected detection of the protein nNOS in the cytoplasm and into miRNAs, we identified 5 sequences as potential targets the nucleus of muscle cells, confirmed that nNOS staining of miR-31, 5 for miR-708, 9 for miR-34c, and 3 for miR- was weaker in the DMDd45-52 compared to the control 212 (Additional file 3: Table S1 and Fig. 3a). Their ability myoblasts (Fig. 4c). Overall, these results were consistent to bind NOS1-3′UTR was then tested in vitro using the with those obtained on BMDd45-55 muscle biopsies, luciferase reporter gene. If a miRNA interacted with namely a higher level of miR-31, miR-708, and miR-34c NOS1-3′UTR, we would measure a decreased luciferase and a decrease in the expression of nNOS, thus allowing signal. Nevertheless, the NOS1-3′UTR being 7165 pb in the use of these DMDd45-52 myoblasts as a suitable in length, it is too large to be fully cloned. Therefore, our vitro cellular model. strategy was to design 4 sequences (parts #1, #2, #3, and To evaluate the effects of the miR-31, miR-708, or #4) which succeed one another with overlapping avoid- miR-34c on the nNOS expression, each of them was ing a miRNA-binding sequence being lost and covering transfected in control myoblasts (Fig. 5a). Overexpres- all the NOS1-3′UTR sequence (Fig. 3a). Each part was sion of the miRNAs was verified by RT-qPCR (Fig. 5a). sub-cloned in a plasmid downstream of the luciferase The location and expression of nNOS protein were first gene, and each of the 4 plasmids was co-transfected in investigated by immunostaining on the transfected myo- HEK293T cells with one candidate or a non-specific blasts (Fig. 5b). Analysis of the pictures showed a de- control miRNA mimic. This strategy would also provide crease of the nNOS labelling in the nuclei of cells a more detailed information about the sequence of overexpressing miR-708 or miR-34c. However, no effect NOS1-3′UTR implicated in miRNA interaction. Our on nNOS expression and location could be observed data showed a significant decrease of luciferase activity when miR-31 was overexpressed compared with myo- when the part #2 was co-transfected with the miR-31 blasts transfected with the non-specific control miRNA. and the part #3 with the miR-708 and when the parts The reduction in the nNOS level was confirmed by #1, or #3, or #4 were co-transfected with the miR-34c. Western blot experiments showing a decrease of about Nevertheless, no decrease of the reporter gene was ob- 30% of nNOS expression in cells overexpressing miR- served when miR-212 was co-transfected with the parts 708 or miR-34c, while no significant decrease could be #1, #2, #3, nor #4. These results demonstrated that observed in overexpressing miR-31 (Fig. 5c). Altogether, miR31, miR-708, and miR34c, but not miR-212, were these results demonstrated that miR-708 or miR-34c able to target NOS1-3′UTR sequences leading to a de- could modulate nNOS expression in human healthy crease of the reporter gene Firefly luciferase expression. myoblasts. Altogether, these results demonstrate that these 4 miR- In DMDd45-52 myoblasts, miR-708 and miR-34c ex- NAs were overexpressed in the muscles of BMDd45-55 pressions increased and nNOS expression decreased Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 7 of 13 Fig. 2 Selection of candidate miRNAs. a In silico screening of miRNAs that could target NOS1 (TargetScan Human and microRNA.org). Candidate miRNAs are underlined. TLDA (Additional file 2,TLDA A2,B2) (b) and individual RT-qPCR (c) values of candidate miRNA expression in healthy subject biopsies (ctrl, black circle) and BMDd45-55 patients with asymptomatic phenotype (gray circle), moderate phenotype (gray square), severe phenotype (gray triangle), or not determined phenotype (gray hexagon); data are normalized on average of control expression. Lines represent average of each group. Individual RT-qPCR data are expressed as relative quantification using miR-30b as normalizer, normalized on average of control expression compared to control myoblasts (Fig. 4); we thus investi- transfected with a non-specific control miRNA (Fig. 6b). gated in these cells the consequences of an inhibition of These results were confirmed by Western blot experi- the miR-708 or the miR-34c by using specific antisense ments that showed a significant increase of 2.2 of nNOS oligonucleotides (antimiR-708 or antimiR-34c) on the expression in cells transfected with antimiR-708 or nNOS expression level (Fig. 6). The inhibition of miR- antimiR-34c (Fig. 6c). 708 or the miR-34c levels by their antimiRNAs was vali- dated by RT-qPCR experiments (Fig. 6a). In these cells, Discussion the nNOS location and expression were also investi- In this study, we used a variety of bioinformatic, molecu- gated. Immunofluorescence experiments showed an in- lar, and cell biological methods to demonstrate the role creased staining in the nuclei of cells in which the miR- of miRNAs in driving nNOS expression. We selected 4 708 or the miR-34c were inhibited compared to cells miRNAs (i.e., miR-31, miR-708, miR-34c, and miR-212) Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 8 of 13 Fig. 3 miR-31, miR-708, and miR-34c targeted 3′UTR sequences of NOS1 gene. a Schematic positions of predicted binding sites by microT-CDS Diana Tools in 4 parts of 3′UTR of NOS1. b Relative luciferase activity of indicated miRNA-transfected cells normalized on luciferase activity in non-- specific miRNA transfected cells (miR-neg). Cells were transfected with part 1, part 2, part 3, or part 4 of NOS1-3′UTR and with either miR-neg con- trol (black bar) or miR of interest (gray bar). *p < 0.05 since they were overexpressed in muscular biopsies of Several studies showed a deregulation of miRNA expres- BMDd45-55 patients compared to healthy subjects or in sion in muscles of DMD or BMD patients [14, 23, 33]or muscular biopsies of patients with severe phenotypes in serum of DMD patients [26, 34]. Eisenberg et al. studied compared to other patients. We then determined, in miRNA profile expression in 10 muscular diseases, and silico, that these miRNAs could target sequences in they showed an upregulation of 5 common miRNAs in NOS1-3′UTR. A luciferase reporter study validated the these diseases . They showed also a particular miRNA targeting of NOS1-3′UTR by miR-31, miR-708, and expression profile shared by DMD patients and severe miR-34c. Finally, we validated the effects of the candi- BMD patients but not with moderate BMD patients. date miRNAs in myoblasts. The experiments were car- Among the selected miRNAs in our study, miR-31 was ried out on myoblasts which were a more homogeneous already shown to be overexpressed in mdx mice and in cell population than those of myotubes, from which we muscular biopsies of DMD patients [14, 23, 35]. We never observed 100% of differentiated myotubes and for found here the same results in muscular biopsies of which efficacy of transfection experiments with miR and BMDd45-55 patients, in DMDd45-52 myoblasts, and in antagomiR was much more effective than on differenti- TA muscle of mdx mice (data not shown). Unlike our ated cells. We thus demonstrated that miR-708 and results, Cacchiarelli and colleagues did not observe an miR-34c could decrease nNOS expression in human increase of miR-31 expression in the biopsies of BMD healthy myoblasts and that their inhibition led to an in- patients. However, no information on the DMD gene crease of this protein in DMDd45-52 human cells. mutations and/or phenotypes was given for the patients Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 9 of 13 Fig. 4 miR-31, miR-708, miR-34c, and nNOS expression in DMDd45-52 myoblasts. a miRNA expression in control human myoblasts and DMDd45- 52 human myoblasts. Graph represents relative quantification of miRNA normalized on SNORD44 expression. miR-708 n = 7, miR-31 n = 7, and miR-34c n =8. b nNOS immunoblot in control and DMDd45-52 cells. GAPDH serves as the loading control. Bar graph shows quantification results average of 8 independent experiments. c Control myoblasts immunolabeled with anti-nNOS (red) antibody, nuclei with Dapi (blue), and imaged by confocal microscopy. Scale bars, 10 μm. Representative of 3 independent experiments. *p ≤ 0.05 included in Cacchiarelli et al.’s study. As we found a Nevertheless, one has to consider that the complex higher expression of miR-31 in severe phenotypes than regulation displayed by non-coding RNAs might be dif- in moderate phenotypes (Fig. 2c), we assume that Cac- ferent according to the studied tissues. chiarelli et al.’s patients had moderate phenotypes and Concerning miR-708 and miR-34c, our results showed therefore might not exhibit a high level of miR-31. The an effect of these two miRNAs on nNOS expression in fact that miR-31 could target nNOS by mRNA decay human healthy and DMDd45-52 myoblasts. miR-708 is was described in human atrial myocytes from patients mostly described in cardiac muscle, where it was pro- with atrial fibrillation . In this study, the precise posed to be involved in myocardium regeneration. In- targeted sequence was identified, and it appears to be deed, its overexpression in newborn rodents leads to the the same that we identified by the system of cloning differentiation of cardiac progenitors to cardiomyocytes NOS1-3′UTR downstream luciferase reporter gene by targeting MAPK14, a cell cycle gene . Otherwise, setup in our study (Fig. 3). Surprisingly, our data re- miR-708 expression is decreased in murine myoblasts at- vealed a slight decrease of nNOS expression by miR-31 rophied by dexamethasone treatment, suggesting that overexpression in control human myoblasts. One rea- miR-708 is involved in muscular development . For son could be the level of miR-31. Indeed, in Reilly et al. miR-34c, several studies described it as overexpressed in ’s work, miR-31-fold increase was 2 × 10 compared to mdx mice and in DMD patients [23, 35]. Our data were control condition whereas in our study, miR-31 in- in the same way as miR-34c is overexpressed in creased by a factor of 4 × 10 (Fig. 5a) and therefore BMDd45-55 muscle biopsies, in DMDd45-52 myoblasts, non-sufficient to exhibit a significant effect. However, and in mdx mice (data not shown). This miRNA was we could not transfect more miR-31 because of dele- shown to be a promoter of differentiation of murine terious effect of transfection on human myoblasts. myoblasts targeting YY1, a transcription factor involved Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 10 of 13 Fig. 5 miR-708 and miR-34c overexpression inhibit nNOS expression in transfected control human myoblasts. a miRNA expression in control human myoblasts transfected with non-specific control miRNA (miR-neg, black bar) or indicated selected miRNA (gray bar). Graph represents average of relative quantification of miRNA normalized on SNORD44 expression of 5 (miR-31) or 3 (miR-708 and miR-34c) independent experi- ments. b Control myoblasts immunolabeled with anti-nNOS (red) antibody, nuclei with Dapi (blue), and imaged by confocal microscopy. Scale bars, 10 μm. Representative of 4 independent experiments. c nNOS immunoblot in transfected control human myoblasts. GAPDH serves as the loading control. Bar graph shows quantification results average of 5 independent experiments. *p ≤ 0.05 in cell proliferation  and of porcine satellite cells by experiments on human muscular biopsy of healthy sub- inhibiting Notch1 signal pathway that is involved in sat- jects showed a major 160 kDa in size nNOS isoform, as ellite cell quiescence . expected but also the 140-kDa isoform (Additional file 4: The present study revealed that the nNOS expression Figure S1). Additionally, we described here that the could be modulated by miR-708 and miR-34c. Our re- nNOS protein was localized in the nuclei of human sults clearly showed their effect at the protein level, al- myoblasts, as shown by immunostaining experiments. though we did not success to detect nNOS mRNA in Western blots carried out on nuclear and cytoplasmic myoblasts to demonstrate also the decay of its transcript. fractions confirmed that 140 kDa nNOS was detected in Interestingly, it should be noted that the isoform of nuclei of control and DMDd45-52 myoblasts (Add- nNOS that was detected in myoblasts by Western blot is itional file 4: Figure S1). Furthermore, a protein of about about 140 kDa in size. In mature skeletal muscle, the 160 kDa in size was only visible in nuclear extracts of nNOSμ, a 165-kDa protein, is the major isoform; it is both types of cells. These data were compared to those linked to dystrophin via its PDZ domain  and thus obtained from immunostaining experiments performed located mainly at the sarcolemma (Additional file 4: on DMD patient muscular biopsies which revealed Figure S1). However, this isoform seems too large to cor- nNOS expression in the nuclei of fibers of DMD muscle respond to the nNOS isoform detected in myoblasts. whereas nNOS is sarcolemmal in control muscle as ex- Another isoform, the nNOSβ which is 136 kDa in size, pected (Additional file 4: Figure S1). Nuclear 160-kDa not displaying the PDZ domain , was described to be nNOS localization has been already described during present in the Golgi apparatus of skeletal muscle fibers C2C12 differentiation; however, authors of this study where it modulates the contractile apparatus or at used a N-terminal nNOS antibody, that did not allow the sarcolemma of mice TA muscles . Western blot the detection of nNOS-β, and therefore a 140 kDa Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 11 of 13 Fig. 6 Inhibition of miR-708 and miR-34c increased nNOS expression in transfected DMDd45-52 human myoblasts. a miRNA expression in DMDd45-52 human myoblasts transfected with control non-specific miRNA (miR-neg, black bar) or indicated selected antimiR (gray bar). Graph represents average of relative quantification of miRNA normalized on SNORD44 expression of 6 (antimiR-708) or 3 (antimiR-34c) independent experiments. b DMDd45-52 myoblasts immunolabeled with anti-nNOS (red) antibody, nuclei with Dapi (blue), and imaged by confocal micros- copy. Scale bars, 10 μm. Representative of 5 independent experiments. c nNOS immunoblot in transfected DMDd45-52 human myoblasts. GAPDH serves as the loading control. Bar graph shows quantification results average of 5 independent experiments. *p ≤ 0.05 nNOS isoform . Our data suggest the presence of an dysregulation in the expression of miR-29a, both of isoform of nNOS not yet described in nuclei of myo- which regulate a dystrophin-nNOS-HDAC2 pathway blasts. At transcriptional level, the precise sequence of a . In the present study, we could not exclude a transcript that encodes a nNOS of 140 kDa in size is not link between nuclear nNOS location, HDAC2 nitrosy- described in databases (i.e., Ensembl.org). The complex- lation, and the modulation of the miR-31, miR-708, ity of the mechanisms modulating NOS1 transcription and/or miR-34c expression. Nevertheless, a study in indicates that the nNOS isoform expressed in myoblasts its own right will be necessary to establish this link. and regulated by miR-34c and miR-708 has not been precisely identified and that information on the tran- Conclusions scriptional regulation of its gene remains to be Altogether, the present work highlights two miRNAs thorough. overexpressed in dystrophic human muscle as modula- The exact role of nNOS in nuclear compartment is tors of nNOS expression. This work could explain some still not well-defined. However, NO production has pathological consequences caused by nNOS deficiency been designated as a key player which mediates epi- (i.e., muscle fatigability due to insufficient vasodilation in genetic changes through the direct control of histone exercise, switch to glycolytic metabolism). In particular, deacetylases (HDACs). Indeed, in the mdx mice de- modification of NOS1 expression has a significant nega- fective for NO pathway, the activity of HDAC2 re- tive impact on dystrophic muscle regenerative capacity sulted to be specifically increased . Profiling of , and it has been shown that treatment with NO do- human DMDd45-52 patient myoblasts confirmed the nors can attenuate atrophy and metabolic changes and dysregulation of miR-1 but also found a significant prevent changes in regulation . We show here that Guilbaud et al. Skeletal Muscle (2018) 8:15 Page 12 of 13 inhibitors of miR-708 and/or miR-34c could also be con- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in sidered as therapeutic targets to rescue these defects by published maps and institutional affiliations. increasing the expression of nNOS. Furthermore, the ex- pression and the sarcolemmal localization of the nNOS Author details Sorbonne Université-UMRS974-Inserm-Institut de Myologie, 105 bd de by interacting with the dystrophin has been shown to be l’Hôpital, 75013 Paris, France. AP-HP, Centre de Référence Maladies crucial for contractile activity and muscular strength re- Neuromusculaire Nord, Est, Ile-de-France, G.H. Pitié-Salpêtrière, F-75013 Paris, covery in the canine DMD model (GRMD) . Thus, a France. Laboratoire de Génétique et Biologie Moléculaire, Hôpital Cochin, Paris, France. Généthon, 1 rue de l’Internationale, 91000 Evry, France. therapeutic strategy combining the inhibition of miR- 708 and miR-34c with the restoration of dystrophin will Received: 26 January 2018 Accepted: 4 April 2018 most likely be a benefit for the improvement of pheno- type of DMD and BMD patients. References 1. Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM. 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Skeletal Muscle – Springer Journals
Published: Apr 27, 2018