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 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: Becker muscular dystrophy (BMD); Duchenne muscular dystrophy (DMD); miRNA; nNOS.

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

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

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 control (black bar) or miR of interest (gray bar). *p < 0.05

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

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 experiments. 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

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 microscopy. 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