MicroRNAs (miRNAs) regulate key biological processes in skeletal muscle, such as muscle development and regeneration. An increasing number of studies also suggest their involvement in muscle disease. These studies support their role as diagnostic and/or prognostic biomarkers as well as therapeutic targets.
A specific group of miRNAs, collectively known as myomiRs, is particularly relevant for skeletal muscle myogenesis. This group includes striated muscle-specific (miR-1, miR-133a, miR-206, miR-208a/b, and miR-499) and muscle-enriched (miR-486) entities. Non-muscle-specific miRNAs are also involved in the regulation of satellite cell quiescence and myoblast proliferation and differentiation, key events in muscle regeneration.
The loss of regenerative potential is characteristic of Duchenne muscular dystrophy (DMD). As explained by Aránega et al in the International Journal of Molecular Sciences, “In this muscle dystrophy, the degenerative processes are associated to the loss of proper muscle regeneration, mainly due to muscle stem cells intrinsic defects related to their activation, proliferation, self-renewal, and differentiation contributing to worsen the dystrophic phenotype.”
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Several studies have highlighted a comprehensive set of dysregulated miRNAs in DMD. These may have diagnostic, prognostic, or therapeutic applicability.
Circulating miRNAs in DMD Diagnosis
Trifunov et al evaluated the serum concentrations of miR-30c, miR-181a, and miR-206 in patients with DMD and Becker muscular dystrophy (BMD) over 4 years. Their study revealed the ability of miR-30c to distinguish DMD patients from healthy individuals, regardless of disease stage. In addition, they showed that miR-206 could distinguish between DMD and BMD phenotypes, also regardless of the disease stage.
Trifunov et al stated, “In the context of DMD may be the most interesting dystromir is miR-206 which mediates the increase of utrophin (utrophin is paralogous to dystrophin) expression in skeletal muscles, thus may serve as a potential therapeutic target for DMD.”
miR-206 belongs to a group of miRNAs called dystromirs, which also includes miR-1, miR-133a, miR-133b, and miR-31. These are muscle-specific entities, and their serum levels are increased in patients with DMD. miR-206 shows nearly 100% specificity and sensitivity in distinguishing DMD patients from healthy individuals. Other studies reported higher levels of miR-206 and other dystromirs in ambulant patients compared to nonambulatory patients, as well as its increase with age in younger patients with DMD, aged 2 to 6 years.
Trifunov et al advised, “We also show that it is important to verify in the long-term setup any candidate for the biomarker as we notice that all 3 miRNAs [miR-30c, miR-181a, and miR-206] show the important extent of both inter- and intrapatient variability.” They add that this “could also be a consequence of their involvement in the different biological processes taking [place] simultaneously.”
Several other miRNAs have shown promising results. Besides miR-206, the serum levels of miR-1 and miR-133 increase with progression of muscle damage and are inversely related to the North Star Ambulatory Assessment score in patients with DMD. miR-26a, miR-222, and miR-378a-5p showed potential in identifying the presence of myocardial scars in patients with DMD.
Mousa et al quantified the plasma levels of miR-499, miR-103a-3p, miR-103a-5p, miR-206, miR-208a, miR-223, and miR-191-5p in families with one member diagnosed with DMD (n=29) and healthy control subjects (n=30). They stated, “Our results suggest that the plasma levels of miRNAs have the capability to diagnose DMD patients and more importantly, miRNAs can be used to identify female carriers.”
In particular, they found that both DMD patients and true carriers (mothers) had significantly upregulated levels of miR-499. Additionally, most (78%) potential carriers (sisters) showed high circulating levels of this miRNA. In contrast, most DMD patients and tested female family members had significantly reduced levels of miR-206 and miR-191-5p. Moreover, miR-103a-5p and miR-208a followed a comparable trend in DMD patients and mothers, and miR-103a-3p showed a modest increase in patients’ families.
Another study that quantified miRNAs in urine showed that ambulatory DMD patients had lower urine levels of miR-29c-3p, whereas nonambulatory DMD patients had lower urine levels of miR-23b-3p and miR-21-5p.
Circulating miRNAs in DMD Progression
With DMD progression, clinical presentation of the disease is even more heterogeneous and may include, for example, progressive loss of cardiac function.
“Finding biomarkers associated with the more progressive symptoms of DMD could mean earlier detection and more proactive treatment of some of these symptoms without performing repeated muscle biopsies on young patients,” Hrach and Mangone wrote.
New Research Sheds Light on Heart Disease Mechanism in DMD
Though less explored, a few miRNAs have been assessed in the context of DMD progression. For instance, female DMD carriers with clinically confirmed cardiomyopathy showed increased levels of miR-26a, miR-206, miR-222, miR-342, miR-378a-3p, and miR-378-5p. In addition, female DMD carriers with functional and/or structural abnormalities of the cardiac muscle had lower levels of miR-29c.
Patients with DMD on a daily steroid regimen have increased levels of miR-1, miR-31, and miR-133b. Steroid treatment aims to delay muscle deterioration. Therefore, these dystromirs may be useful as a measure of the remaining muscle mass in DMD patients with progressive phenotypes.
miRNAs With Therapeutic Potential in DMD
Among the most promising miRNAs with therapeutic potential in DMD are miR-21, miR-29, miR-31, and miR-206.
Inhibition of miR-21 in mdx mice reduced fibrosis in the diaphragm and improved muscle
homeostasis. Aránega et al explained, “Treatment by using miR-21 inhibitors would reduce fibrosis in DMD, slowing down disease progression and thus facilitating the period for muscle regeneration treatment therapies.”
Intramuscular and/or intravenous injections of miR-29 in mdx mice promoted muscle function and inhibited fibrogenesis. In addition, its overexpression along with micro-dystrophin gene delivery in a mouse model with a more severe dystrophic phenotype suppressed fibrosis, restored muscle function, and increased absolute and specific forces.
The combination of exon skipping with miR-31 inhibition in human DMD myoblasts increased dystrophin
synthesis. According to Aránega et al, “Since miR-31 also targets myogenic factor Myf5 and its downregulation leads to [satellite cell] activation and differentiation, we can speculate that the inhibition of miR-31 could be a strong candidate to be proposed as a therapeutic tool to improve both muscle differentiation and dystrophin restoration.”
Muscular inhibition of miR-206 in mdx mice increased the expression of compensatory “booster genes” (eg, vascular endothelial growth factor A [VEGFA] and utrophin), thereby improving motor function and reducing muscle fibrosis.
Despite the recent advances in DMD-related miRNA investigation, Hrach and Mangone advise that “the circumstances under which miRNAs can and should be administered therapeutically [require] careful consideration.”
DMD symptoms and disease progression vary from patient to patient, and the expression and function of miRNAs are tissue-specific. Therefore, miRNAs may carry out different functions in different tissues, and a given treatment option may not fit all realities.
References
Aránega AE, Lozano-Velasco E, Rodriguez-Outeiriño L, Ramírez de Acuña F, Franco D, Hernández-Torres F. MiRNAs and muscle regeneration: therapeutic targets in Duchenne muscular dystrophy. Int J Mol Sci. 2021;22(8):4236. doi:10.3390/ijms22084236
Hrach HC, Mangone M. miRNA profiling for early detection and treatment of Duchenne muscular dystrophy. Int J Mol Sci. 2019;20(18):4638. doi:10.3390/ijms20184638
Trifunov S, Natera-de Benito D, Exposito Escudero JM, et al. Longitudinal study of three microRNAs in Duchenne muscular dystrophy and Becker muscular dystrophy. Front Neurol. 2020;11:304. doi:10.3389/fneur.2020.00304
Mousa NO, Abdellatif A, Fahmy N, Zada S, El-Fawal H, Osman A. Circulating microRNAs in Duchenne muscular dystrophy. Clin Neurol Neurosurg. 2020;189:105634. doi:10.1016/j.clineuro.2019.105634
Rybalka E, Timpani CA. Micro (mRNA) molecules could pack a big punch in the fight against neuromuscular disease. J Physiol. 2021;599(1):5-6. doi:10.1113/JP280872
