Muscle-specific miRNAs Research Articles

Circulating microRNAs after a 24-h ultramarathon run in relation to muscle damage markers in elite athletes.

Ultra-endurance sports are growing in popularity but can be associated with adverse health effects, such as exercise-induced muscle damage (EIMD), which can lead to exertional rhabdomyolysis. Circulating microRNAs (miRNAs) may be useful to approach the degree of EIMD. We aimed to (1) investigate the relevance of circulating miRNAs as biomarkers of muscle damage and (2) examine the acute response of skeletal/cardiac muscle and kidney biomarkers to a 24-h run in elite athletes. Eleven elite athletes participated in the 24-h run World Championships. Counter-movement jump (CMJ), creatine kinase (CK), myoglobin (Mb), creatinine (Cr), high-sensitive cardiac troponin T (hs-cTnT), and muscle-specific miRNA (myomiR) levels were measured before, immediately after, and 24 and 48h after the race. CMJ height was reduced immediately after the race (-84.0±25.2%, p<0.001) and remained low at 24h (-43.6±20.4%, p=0.002). We observed high CK activity (53239±63608 U/L, p<0.001) immediately after the race, and it remained elevated 24h after (p<0.01). Circulating myomiR levels (miR-1-3p, miR-133a-3p, miR-133b, miR-208a-3p, miR-208b-3p, and miR-499a-5p) were elevated immediately after the 24-h run (fold changes: 18-124,723, p<0.001) and significantly (p<0.05) correlated or tended to significantly (p<0.07) correlate with the reduction in CMJ height at 24h. We found no significant correlation between CMJ height loss at 24h and CK (p=0.23) or Mb (p=0.41) values. All elite ultramarathon runners included in our study were diagnosed with exertional rhabdomyolysis after the 24-h ultramarathon race. MyomiR levels may be useful to approach the degree of muscle damage.

METTL3 regulates skeletal muscle specific miRNAs at both transcriptional and post-transcriptional levels

METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations.

MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

Myocyte enhancer factor 2C (MEF2C) is a transcription factor that regulates heart and skeletal muscle differentiation and growth. Several protein-encoding genes were identified as targets of this factor; however, little is known about its contribution to the microtranscriptome composition and dynamics in myogenic programs. In this report, we aimed to address this question. Deep sequencing of small RNAs of human muscle cells revealed a set of microRNAs (miRNAs), including several muscle-specific miRNAs, that are sensitive to MEF2C depletion. As expected, in cells with knockdown of MEF2C, we found mostly downregulated miRNAs; nevertheless, as much as one-third of altered miRNAs were upregulated. The majority of these changes are driven by transcription efficiency. Moreover, we found that MEF2C affects nontemplated 3′-end nucleotide addition of miRNAs, mainly oligouridylation. The rate of these modifications is associated with the level of TUT4 which mediates RNA 3′-uridylation. Finally, we found that a quarter of miRNAs which significantly changed upon differentiation of human skeletal myoblasts is inversely altered in MEF2C deficient cells. We concluded that MEF2C is an essential factor regulating both the quantity and quality of the microtranscriptome, leaving an imprint on the stability and perhaps specificity of many miRNAs during the differentiation of muscle cells.

The functional analysis of transiently upregulated miR-101 suggests a "braking" regulatory mechanism during myogenesis.

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and microRNAs (miRNAs). A group of muscle-specific miRNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth. However, the functional role and regulatory mechanism of most non-muscle-specific miRNAs with stage-specific changes during differentiation are largely unclear. Here, we describe the functional characterization of miR-101a/b, a pair of non-muscle-specific miRNAs that show the largest change among a group of transiently upregulated miRNAs during myogenesis in C2C12 cells. The overexpression of miR-101a/b inhibits myoblast differentiation by suppressing the p38/MAPK, Interferon Gamma, and Wnt pathways and enhancing the C/EBP pathway. Mef2a, a key protein in the p38/MAPK pathway, was identified as a direct target of miR-101a/b. Interestingly, we found that the long non-coding RNA (lncRNA) Malat1, which promotes muscle differentiation, interacts with miR-101a/b, and this interaction competes with Mef2a mRNA to relieve the inhibition of the p38/MAPK pathway during myogenesis. These results uncovered a "braking" role in differentiation of transiently upregulated miRNAs and provided new insights into the competing endogenous RNA (ceRNA) regulatory mechanism in myoblast differentiation and myogenesis.

Muscle-derived exosomes encapsulate myomiRs and are involved in local skeletal muscle tissue communication.

Exosomes are extracellular vesicles that are released from most cell types encapsulating specific molecular cargo. Exosomes serve as mediators of cell-to-cell and tissue-to-tissue communications under normal and pathological conditions. It has been shown that exosomes carrying muscle-specific miRNAs, myomiRs, are secreted from skeletal muscle cells in vitro and are elevated in the blood of muscle disease patients. The aim of this study was to investigate the secretion of exosomes encapsulating the four myomiRs from skeletal muscle tissues and to assess their role in inter-tissue communication between neighboring skeletal muscles in vivo. We demonstrate, for the first time, that isolated, intact skeletal muscle tissues secrete exosomes encapsulating the four myomiRs, miR-1, miR-133a, miR-133b, and miR-206. Notably, we show that the sorting of the four myomiRs within exosomes varies between skeletal muscles of different muscle fiber-type composition. miR-133a and miR-133b downregulation in TA muscles caused a reduction of their levels in neighboring skeletal muscles and in serum exosomes. In conclusion, our results reveal that skeletal muscle-derived exosomes encapsulate the four myomiRs, some of which enter the blood, while a portion is used for the local communication between proximal muscle tissues. These findings provide important evidence regarding novel pathways implicated in skeletal muscle function.

LncRNAs and their regulatory networks in breast muscle tissue of Chinese Gushi chickens during late postnatal development

BackgroundChicken skeletal muscle is an important economic product. The late stages of chicken development constitute the main period that affects meat production. LncRNAs play important roles in controlling the epigenetic process of growth and development. However, studies on the role of lncRNAs in the late stages of chicken breast muscle development are still lacking. In this study, to investigate the expression characteristics of lncRNAs during chicken muscle development, 12 cDNA libraries were constructed from Gushi chicken breast muscle samples from 6-, 14-, 22-, and 30-week-old chickens.ResultsA total of 1252 new lncRNAs and 1376 annotated lncRNAs were identified. Furthermore, 53, 61, 50, 153, 117, and 78 DE-lncRNAs were found in the W14 vs. W6, W22 vs. W14, W22 vs. W6, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparison groups, respectively. After GO enrichment analysis of the DE-lncRNAs, several muscle development-related GO terms were found in the W22 vs. W14 comparison group. Moreover, it was found that the MAPK signaling pathway was one of the most frequently enriched pathways in the different comparison groups. In addition, 12 common target DE-miRNAs of DE-lncRNAs were found in different comparison groups, some of which were muscle-specific miRNAs, such as gga-miR-206, gga-miR-1a-3p, and miR-133a-3p. Interestingly, the precursors of four newly identified miRNAs were found to be homologous to lncRNAs. Additionally, we found some ceRNA networks associated with muscle development-related GO terms. For example, the ceRNA networks contained the DYNLL2 gene with 12 lncRNAs that targeted 2 miRNAs. We also constructed PPI networks, such as IGF-I-EGF and FZD6-WNT11.ConclusionsThis study revealed, for the first time, the dynamic changes in lncRNA expression in Gushi chicken breast muscle at different periods and revealed that the MAPK signaling pathway plays a vital role in muscle development. Furthermore, MEF2C and its target lncRNA may be involved in muscle regulation through the MAPK signaling pathway. This research provided valuable resources for elucidating posttranscriptional regulatory mechanisms to promote the development of chicken breast muscles after hatching.

Therapeutic potential of miRNAs in Duchenne muscular dystrophy and other neuromuscular diseases.

Therapeutic potential of miRNAs in Duchenne muscular dystrophy and other neuromuscular diseases.

MiR-206 enforces a slow muscle phenotype.

Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms.

Expression Analysis of Muscle-Specific miRNAs in Plasma-Derived Extracellular Vesicles from Patients with Chronic Obstructive Pulmonary Disease.

MicroRNAs (miRNAs) are a class of short non-coding RNAs involved in the regulation of gene expression and the control of several cellular processes at physiological and pathological levels. Furthermore, extracellular vesicles (EV), which are small membrane-bound vesicles secreted by cells in the extracellular environment, contain functional miRNAs. The remarkable deregulation of many miRNAs has been demonstrated in respiratory diseases. Among them, miR-206, miR-133a-5p, and miR-133a-3p are striated muscle-specific miRNAs (myo-miRNA), related to skeletal muscle dysfunction, one of the commonest systemic manifestations in patients with chronic obstructive pulmonary disease (COPD). Nevertheless, their circulating expression in COPD patients is not demonstrated. For these reasons, we performed a pilot study to analyze the expression profiles of myo-miRNAs in plasma-derived EV from patients with COPD. We analyzed the expression profiles of selected myo-miRNAs in plasma-derived EV from COPD. Receiver operating characteristic analyses were carried out to evaluate whether selected plasma miRNAs were able to discriminate between different groups of COPD patients. We found EV-embedded myo-miRNAs in the bloodstream of COPD patients. Specifically, miR-206, miR-133a-5p and miR-133a-3p were significantly upregulated in group B patients. Receiver operating characteristic analyses of the combination of these selected miRNAs showed their high capacity to discriminate group B from other COPD patients. Our data provide evidence that myo-miRNA are present in EV in the plasma of COPD patients and their expression (miR-206, miR-133a-5p, and miR-133a-3p) can discriminate group B from group C patients. The future analysis of a larger number of patients should allow us to obtain more refined correlations.

Transcriptome Characterization of Repressed Embryonic Myogenesis Due to Maternal Calorie Restriction.

Fetal malnutrition decreases skeletal myofiber number and muscle mass in neonatal mammals, which increases the risk of developing obesity and diabetes in adult life. However, the associated molecular mechanisms remain unclear. Here, we investigated how the nutrient (calorie) availability affects embryonic myogenesis using a porcine model. Sows were given a normal or calorie restricted diet, following which skeletal muscle was harvested from the fetuses at 35, 55, and 90 days of gestation (dg) and used for histochemical analysis and high-throughput sequencing. We observed abrupt repression of primary myofiber formation following maternal calorie restriction (MCR). Transcriptome profiling of prenatal muscles revealed that critical genes and muscle-specific miRNAs associated with increased proliferation and myoblast differentiation were downregulated during MCR-induced repression of myogenesis. Moreover, we identified several novel miRNA-mRNA interactions through an integrative analysis of their expression profiles, devising a putative molecular network involved in the regulation of myogenesis. Interestingly, NC_010454.3_1179 was identified as a novel myogenic miRNA that can base-pair with sequences in the 3′-UTR of myogenic differentiation protein 1 (MyoD1). And we found that this UTR inhibited the expression of a linked reporter gene encoding a key myogenic regulatory factor, resulting in suppression of myogenesis. Our results greatly increase our understanding of the mechanisms underlying the nutrient-modulated myogenesis, and may also serve as a valuable resource for further investigation of fundamental developmental processes or assist in rational target selection ameliorating repressed myogenesis under fetal malnutrition.

Oncolytic Activity of Targeted Picornaviruses Formulated as Synthetic Infectious RNA

Infectious nucleic acid has been proposed as a superior formulation for oncolytic virus therapy. Oncolytic picornaviruses can be formulated as infectious RNA (iRNA), and their unwanted tropisms eliminated by microRNA (miRNA) detargeting. However, genomic insertion of miRNA target sequences into coxsackievirus A21 (CVA21) iRNA compromised its specific infectivity, negating further development as a novel oncolytic virus formulation. To address this limitation, we substituted a muscle-specific miRNA response element for the spacer region downstream of the internal ribosomal entry site in the 5′ non-coding region of CVA21 iRNA, thereby preserving genome length while avoiding the disruption of known surrounding RNA structural elements. This new iRNA (R-CVA21) retained high specific infectivity, rapidly generating replicating miRNA-detargeted viruses following transfection in H1-HeLa cells. Further, in contrast with alternatively configured iRNAs that were tested in parallel, intratumoral administration of R-CVA21 generated a spreading oncolytic infection that was curative in treated animals without associated myotoxicity. Moreover, R-CVA21 also exhibited superior miRNA response element stability in vivo. This novel formulation is a promising agent for clinical translation.

MicroRNAs and HDAC4 protein expression in the skeletal muscle of ALS patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive degeneration of motor neurons. MicroRNAs are 17-27 nucleotide long molecules that regulate post-transcriptional mRNA expression. The aim of this study was to investigate the role of microRNAs in the skeletal muscle of ALS patients and correlate these results with the expression of histone deacetylase 4 (HDAC4) protein. We measured the expression levels of muscle-specific microRNAs (miR-1, miR-133a, miR-133b, miR-206), inflammatory micro-RNAs (miR-27a, miR-221, miR-155), and HDAC4 protein content on western blotting in muscle biopsies obtained for diagnostic reasons in 18 ALS patients: 8 genetic forms (C9-ALS and SOD1-ALS), 5 sporadic cases (SALS), and 5 ALS cases affected only by upper motor neuron disease (UMN). In muscle of patients with genetic forms of ALS, we found a strong upregulation of miR-206, a muscle-specific miRNA involved in neuromuscular junction (NMJ), regeneration and muscle atrophy, and a decreased expression of HDAC4 protein levels, which is involved both in denervation and regulation of miR-206 in ALS pathophysiology. In these patients, we also observed an increase of inflammatory miRNAs. The different expression of miRNAs and HDAC4 in genetic ALS vs. SALS and UMN cases is likely to be correlated to different pathogenic mechanisms.

Regulation of Circulatory Muscle-specific MicroRNA during 8 km Run.

Acute prolonged endurance running has been shown to alter muscle-specific circulating microRNA (miRNA) levels. Here, eighteen participants completed an 8 km run. We assessed the levels of hsa-miR-1-3p, -133a-3p, -133b, and -206 and their correlation with conventional biomarkers following exercise. Compared to before exercise (Pre), 8 km run significantly increased the lactate level immediately after exercise (0 h). Myoglobin (Mb) level increased at 0 h while creatine kinase (CK) level increased 24 h after exercise (24 h). The levels of creatine kinase MB isoenzyme (CK-MB) and cardiac troponin I (cTnI) were all elevated at 24 h and within the normal physiological range; The levels of hsa-miR-1-3p, -133a-3p, -133b significantly increased at 0 h but only hsa-miR-133a-3p still elevated at 24 h. Only hsa-miR-206 level decreased at 24 h; Additionally, the changes of hsa-miR-1-3p and hsa-miR-133a-3p were correlated with Mb at 24 h. These findings suggest that muscle-specific miRNA elevation in plasma is likely physiological and that these miRNA may be used as potential biomarkers for load monitoring in individuals.

Correlation between ETFDH mutations and dysregulation of serum myomiRs in MADD patients.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare fatty acids oxidation disorder which is often associated with deficiency of electron transfer flavoprotein dehydrogenase (ETFDH). In this study we reported clinical features and evaluation of expression profile of circulating muscle-specific miRNAs (myomiRs) in two MADD patients carrying different ETFDH gene mutations. Patient 1 was a compound heterozygote for two missense mutations. She showed a late onset MADD clinical phenotype and a significant increase of serum myomiRs. Patient 2, carrying a missense and a frameshift mutation, displayed early onset symptoms and a slight increase of some serum myomiRs.

MiR-206 knockout shows it is critical for myogenesis and directly regulates newly identified target mRNAs

ABSTRACT The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six of these targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.

Poly(lactic-co-glycolic acid)/Polyethylenimine Nanocarriers for Direct Genetic Reprogramming of MicroRNA Targeting Cardiac Fibroblasts

Cardiovascular disease remains a major cause of deaths globally. Post-heart-infarction, the most abundant cell type of heart, ‘fibroblasts’ undergoes a series of culminating events that lead to fibrotic scar tissue. In many organisms, injury to the heart can be restored but the adult human heart is unable to efficiently regenerate after ischemic injury. So, the inefficiency of heart to regenerate on its own after ischemic injury accounts for its reprogramming. Herein, we demonstrate the effect of microRNAs encapsulating Poly (lactic-co-glycolic acid) (PLGA)-Polyethylenimine (PEI) nanocarriers for direct reprogramming of cardiac fibroblast to cardiomyocyte-like cells. Dual, cardiac muscle specific miRNA (miR-1 and miR-133a) polyplexes were encapsulated in biodegradable PLGA nanospheres. Cytocompatibility of the nanocomplexes were evaluated by various in vitro assays, confirming their safety profile. The change in cardiac fibroblast phenotype to cardiomyocyte was identified by the expression of late-stage s...

Circulating MyomiRs as Potential Biomarkers to Monitor Response to Nusinersen in Pediatric SMA Patients.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in survival motor neuron (SMN) 1 gene, resulting in a truncated SMN protein responsible for degeneration of brain stem and spinal motor neurons. The paralogous SMN2 gene partially compensates full-length SMN protein production, mitigating the phenotype. Antisense oligonucleotide nusinersen (Spinraza®) enhances SMN2 gene expression. SMN is involved in RNA metabolism and biogenesis of microRNA (miRNA), key gene expression modulators, whose dysregulation contributes to neuromuscular diseases. They are stable in body fluids and may reflect distinct pathophysiological states, thus acting as promising biomarkers. Muscle-specific miRNAs (myomiRs) as biomarkers for clinical use in SMA have not been investigated yet. Here, we analyzed the expression of miR-133a, -133b, -206 and -1, in serum of 21 infantile SMA patients at baseline and after 6 months of nusinersen treatment, and correlated molecular data with response to therapy evaluated by the Hammersmith Functional Motor Scale Expanded (HFMSE). Our results demonstrate that myomiR serological levels decrease over disease course upon nusinersen treatment. Notably, miR-133a reduction predicted patients’ response to therapy. Our findings identify myomiRs as potential biomarkers to monitor disease progression and therapeutic response in SMA patients.

Biomechanical effects on microRNA expression in skeletal muscle differentiation

We examined whether mechanical stretch affected expression of muscle-specific microRNAs (miRNAs) that regulate proliferation (miR-133a) or differentiation (miR-1, -206). Real-time quantitative RT-PCR was used to assess miRNA regulation in the murine myoblast cell line C2C12 exposed to stretch regimens that promote either proliferation (high stretch: 17%, 1 Hz) or differentiation (moderate stretch: 10%, 0.5 Hz) after adding media that promotes differentiation. Controls consisted of myoblasts cultured under static conditions. While miRNA expression was not affected by high stretch, a significant effect of stretch (P

Time-Course Responses of Muscle-Specific MicroRNAs Following Acute Uphill or Downhill Exercise in Sprague-Dawley Rats.

Objective: The physiological characteristics and acute responses underpinning uphill running differ from those of downhill running and remain less understood. This study aimed to evaluate time-course changes of muscle-specific microRNA (miRNA) responses in striated muscle or circulation in response to uphill and downhill running.Methods: Male Sprague-Dawley rats (n = 84) were randomly assigned to a sedentary group (n = 12) and an exercise group (n = 72). The exercise group performed 90 min of uphill or downhill running. The striated muscle (quadriceps, gastrocnemius, soleus, and cardiac muscle) or circulation (plasma, exosome, exosome-free) levels of six muscle-specific miRNAs (miR-1, miR-133a, miR-133b, miR-206, miR-208a, and miR-499) were assessed at rest, immediately following exercise, and during recovery (1 h and 48 h).Results: Our results show that miR-1 and miR-133a levels are both decreased in quadriceps following downhill running (p < 0.05) while there is no change after uphill running (p > 0.05). In gastrocnemius, both uphill and downhill running decreased miR-1 level immediately after exercise and returned to baseline during recovery (p < 0.05): interestingly, only miR-499 significantly increased following uphill running (p > 0.05). Of the cell-free miRNAs in circulation, only the miR-133b levels in plasma were not affected following uphill running (p > 0.05); the other miRNA levels significantly increased immediately after exercise (p < 0.05), decreased at 1 h and significantly increased at 48 h after exercise (p < 0.05). All selected miRNA levels in exosomes were not affected following uphill running (p > 0.05), while all selected miRNA levels significantly increased during early recovery after downhill running (p > 0.05). In addition, only the miR-133a level in the exosome-free condition showed significant changes following uphill running (p < 0.05), while miR-1, miR-133a, and miR-499 levels showed significant changes after downhill running (p < 0.05).Conclusion: The results indicate that miRNA undergoes dynamic changes in tissue may play an important role in regulating different stress/adaptation following uphill and downhill running. It is likely that changed miRNA levels in plasma may act as a new biomarker for monitoring whole muscular stress during recovery.

P.144A cross sectional and longitudinal miRNA profiling study identified a set of novel free-circulating and exosomal miRNAs dysregulated in plasma from Duchenne muscular dystrophy patients

There is a growing need for accurate, reliable and minimally-invasive biomarkers for monitoring DMD progression and response to treatment. microRNAs (miRNAs) are an attractive disease biomarker candidate due to their straightforward isolation from biofluids and known dysregulation in DMD patients. Previous cross-sectional studies identified dysregulation of several muscle-specific miRNAs in DMD patients. However, to date, no longitudinal studies have been performed. We performed the first miRNA profiling studies on free-circulating (fc) and exosomal (ex) miRNAs isolated from DMD patients' plasma, aiming to find novel cross-sectionally and longitudinally dysregulated miRNAs. Plasma was collected from 14 controls, 29 ambulant (A) and 17 non-ambulant (NA) DMD patients. Longitudinal fc-miRNA expression patterns were evaluated between two time-points in ambulant (n=7) and non-ambulant (n=2) DMD patients. MiRNA profiling and validation studies revealed longitudinal upregulation of fc-miR-1-3p and miR-122-5p in A DMD patients compared to controls. Cross-sectional analysis revealed one novel fc-miRNA and two novel ex-miRNAs significantly upregulated in DMD compared to controls, and a novel miRNA dysregulated between A and NA DMD patients. Dysregulated miRNAs significantly correlated with clinical functional tests, including ex-miR-29 with several lower limb measures and ex-miR-33a-5p with grip strength. Additionally, miR-29c-3p expression (fc & ex) was significantly downregulated in patients taking intermittent corticosteroids compared to controls. We confirmed the dysregulation of previously-published miRNAs and five novel free-circulating and exosomal miRNAs in DMD patients. MiR-29c-3p, a fibrosis-regulator, was down-regulated in DMD patients, exosomal expression correlated with functional measures and its levels were also modulated by daily corticosteroid therapy. Therefore, miR-29-3p is a promising biomarker in DMD.