Comprehensive small RNA sequencing analysis in dystrophic serum and muscle. Mus musculus

Extracellular small RNAs (sRNAs), such as microRNAs (miRNAs), are promising minimally-invasive biomarkers for a variety of disease conditions, including the congenital muscle-wasting disorder Duchenne Muscular Dystrophy (DMD). To identify novel sRNA DMD biomarkers, and to investigate the relationship between serum and muscle miRNA levels, we performed sRNA sequencing in serum and four muscle tissues taken from wild-type, dystrophic mdx mice, and mdx mice treated with antisense oligonucleotides which restore dystrophin protein expression by exon skipping. Using this approach, we identified differentially abundant miRNAs in dystrophic serum, and a set of eight miRNAs that were commonly up-regulated in all mdx tissues analyzed. One of these miRNAs, miR-483-3p, was elevated in dystrophic serum and muscle. Exon skipping treatment induced a shift in miRNA levels (including miR-483-3p) towards wild-type levels in mdx serum. In contrast, the dystrophic miRNome was only partially restored in diaphragm, gastrocnemius and soleus, and essentially unchanged in tibialis anterior. In humans the pre-miR-483 strand processing bias is reversed, and accordingly miR-483-5p was found to be significantly elevated in DMD patient serum. Absolute miRNA expression in muscle was positively correlated with abundance in serum, although multiple highly expressed miRNAs in muscle were unchanged, or exhibited reduced abundance levels, in mdx serum. These findings suggest that passive leakage from damaged muscle alone is insufficient to explain elevated miRNA levels in dystrophic serum, and that miRNA release is likely to be selective in certain cases. Analysis of other non-coding RNAs revealed differentially abundant extracellular tRNA-fragments, snRNA-derived species, and piRNA-like sRNAs. Interestingly, many of these non-coding RNA species were also detectable in muscle. In conclusion, this study has identified novel DMD biomarkers and revealed new insights into the biology of extracellular sRNAs in dystrophic muscle.