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Extracellular microRNAs are dynamic non-vesicular biomarkers of muscle turnover.

Roberts TC, Godfrey C, McClorey G, Vader P, Briggs D, Gardiner C, Aoki Y, Sargent I, Morgan JE, Wood MJ - Nucleic Acids Res. (2013)

Bottom Line: Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature.Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes.In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA, Dubowitz Neuromuscular Centre, UCL Institute of Child Health, 30 Guildford Street, London, WC1N 1EH, UK and Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK.

ABSTRACT
Extracellular microRNAs (miRNAs) are promising biomarkers of the inherited muscle wasting condition Duchenne muscular dystrophy, as they allow non-invasive monitoring of either disease progression or response to therapy. In this study, serum miRNA profiling reveals a distinct extracellular miRNA signature in dystrophin-deficient mdx mice, which shows profound dose-responsive restoration following dystrophin rescue. Extracellular dystrophy-associated miRNAs (dystromiRs) show dynamic patterns of expression that mirror the progression of muscle pathology in mdx mice. Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature. Similarly, extracellular dystromiRs were elevated following experimentally-induced skeletal muscle injury and regeneration in non-dystrophic mice. Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes. In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

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Related in: MedlinePlus

Identification of novel extracellular biomarkers by serum miRNA profiling. Serum from C57Bl/10, mdx and Pip6a-PMO-treated mdx mice was analysed by miRCURY LNA SYBR Green miRNA RT-qPCR array. (A) Heatmap of relative expression values for all miRNAs detected in all samples. Red indicates higher abundance, and blue indicates lower abundance. Scale bar values represent ΔΔCt values. The heatmap was produced by median centering expression values for each miRNA and then performing hierarchical clustering. (B) Plot of P-value versus fold change for miRNAs detected in all samples.
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gkt724-F2: Identification of novel extracellular biomarkers by serum miRNA profiling. Serum from C57Bl/10, mdx and Pip6a-PMO-treated mdx mice was analysed by miRCURY LNA SYBR Green miRNA RT-qPCR array. (A) Heatmap of relative expression values for all miRNAs detected in all samples. Red indicates higher abundance, and blue indicates lower abundance. Scale bar values represent ΔΔCt values. The heatmap was produced by median centering expression values for each miRNA and then performing hierarchical clustering. (B) Plot of P-value versus fold change for miRNAs detected in all samples.

Mentions: To identify novel differentially expressed serum dystromiRs, miRNA expression profiling was performed by miRCURY LNA SYBR green RT-qPCR array. The 12-week-old male mdx animals (n = 4) were treated with a single intravenous injection of Pip6a-PMO (12.5 mg/kg) and sacrificed 2 weeks later. Serum samples from these mice were compared with age- and sex-matched C57Bl/10 (wild-type) and untreated mdx serum. In all, 123 miRNAs were detected in all samples. The number of miRNAs detected in each sample varied from 168 to 258. Experimental groups clustered as expected when analysed by hierarchical clustering (Figure 2A) or principal component analysis (Supplementary Figure S1): treated samples clustered with the C57Bl/10 wild-type controls and away from the mdx samples. Fifteen miRNAs were significantly changed at the P < 0.01 level, and a further 42 miRNAs were significantly changed at the P < 0.05 level (one-way ANOVA). The abundance of all of these 57 miRNAs increased in mdx serum and was restored in the Pip6a-PMO-treated samples, suggesting a profound restoration of the circulating miRNA profile following treatment (Figure 2B). Aside from the established dystromiRs, the four most differentially abundant miRNAs were miR-22, miR-30a, miR-193b and miR-378 (Figure 2A). These candidate miRNAs were selected for further evaluation as putative biomarkers.Figure 2.


Extracellular microRNAs are dynamic non-vesicular biomarkers of muscle turnover.

Roberts TC, Godfrey C, McClorey G, Vader P, Briggs D, Gardiner C, Aoki Y, Sargent I, Morgan JE, Wood MJ - Nucleic Acids Res. (2013)

Identification of novel extracellular biomarkers by serum miRNA profiling. Serum from C57Bl/10, mdx and Pip6a-PMO-treated mdx mice was analysed by miRCURY LNA SYBR Green miRNA RT-qPCR array. (A) Heatmap of relative expression values for all miRNAs detected in all samples. Red indicates higher abundance, and blue indicates lower abundance. Scale bar values represent ΔΔCt values. The heatmap was produced by median centering expression values for each miRNA and then performing hierarchical clustering. (B) Plot of P-value versus fold change for miRNAs detected in all samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814379&req=5

gkt724-F2: Identification of novel extracellular biomarkers by serum miRNA profiling. Serum from C57Bl/10, mdx and Pip6a-PMO-treated mdx mice was analysed by miRCURY LNA SYBR Green miRNA RT-qPCR array. (A) Heatmap of relative expression values for all miRNAs detected in all samples. Red indicates higher abundance, and blue indicates lower abundance. Scale bar values represent ΔΔCt values. The heatmap was produced by median centering expression values for each miRNA and then performing hierarchical clustering. (B) Plot of P-value versus fold change for miRNAs detected in all samples.
Mentions: To identify novel differentially expressed serum dystromiRs, miRNA expression profiling was performed by miRCURY LNA SYBR green RT-qPCR array. The 12-week-old male mdx animals (n = 4) were treated with a single intravenous injection of Pip6a-PMO (12.5 mg/kg) and sacrificed 2 weeks later. Serum samples from these mice were compared with age- and sex-matched C57Bl/10 (wild-type) and untreated mdx serum. In all, 123 miRNAs were detected in all samples. The number of miRNAs detected in each sample varied from 168 to 258. Experimental groups clustered as expected when analysed by hierarchical clustering (Figure 2A) or principal component analysis (Supplementary Figure S1): treated samples clustered with the C57Bl/10 wild-type controls and away from the mdx samples. Fifteen miRNAs were significantly changed at the P < 0.01 level, and a further 42 miRNAs were significantly changed at the P < 0.05 level (one-way ANOVA). The abundance of all of these 57 miRNAs increased in mdx serum and was restored in the Pip6a-PMO-treated samples, suggesting a profound restoration of the circulating miRNA profile following treatment (Figure 2B). Aside from the established dystromiRs, the four most differentially abundant miRNAs were miR-22, miR-30a, miR-193b and miR-378 (Figure 2A). These candidate miRNAs were selected for further evaluation as putative biomarkers.Figure 2.

Bottom Line: Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature.Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes.In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA, Dubowitz Neuromuscular Centre, UCL Institute of Child Health, 30 Guildford Street, London, WC1N 1EH, UK and Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK.

ABSTRACT
Extracellular microRNAs (miRNAs) are promising biomarkers of the inherited muscle wasting condition Duchenne muscular dystrophy, as they allow non-invasive monitoring of either disease progression or response to therapy. In this study, serum miRNA profiling reveals a distinct extracellular miRNA signature in dystrophin-deficient mdx mice, which shows profound dose-responsive restoration following dystrophin rescue. Extracellular dystrophy-associated miRNAs (dystromiRs) show dynamic patterns of expression that mirror the progression of muscle pathology in mdx mice. Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature. Similarly, extracellular dystromiRs were elevated following experimentally-induced skeletal muscle injury and regeneration in non-dystrophic mice. Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes. In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

Show MeSH
Related in: MedlinePlus