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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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Time course of serum dystromiR abundance. Male C57Bl/10, mdx and Pip6e-PMO-treated mdx mice were sacrificed at various ages, and serum miRNA levels were determined by small RNA TaqMan RT-qPCR. (A) miR-1, (B) miR-133a and (C) miR-206 abundance was normalized to an external spike-in control. All miRNA expression data were normalized to the mean of the 8-week-old C57Bl/10 group. Arrow indicates time of injection (single intravenous 12.5 mg/kg dose Pip6e-PMO). The time course of exon skipping and dystrophin restoration was also determined in the TA of the treated mdx samples at various time points by (D) quantitative immunofluorescence and (E) RT-qPCR, respectively. Values are mean ± SEM, n = 3–8.
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gkt724-F3: Time course of serum dystromiR abundance. Male C57Bl/10, mdx and Pip6e-PMO-treated mdx mice were sacrificed at various ages, and serum miRNA levels were determined by small RNA TaqMan RT-qPCR. (A) miR-1, (B) miR-133a and (C) miR-206 abundance was normalized to an external spike-in control. All miRNA expression data were normalized to the mean of the 8-week-old C57Bl/10 group. Arrow indicates time of injection (single intravenous 12.5 mg/kg dose Pip6e-PMO). The time course of exon skipping and dystrophin restoration was also determined in the TA of the treated mdx samples at various time points by (D) quantitative immunofluorescence and (E) RT-qPCR, respectively. Values are mean ± SEM, n = 3–8.

Mentions: To investigate how serum miRNA levels change during disease progression or in response to dystrophin restoration C57Bl/10, mdx and mdx mice treated with Pip6e-PMO were sacrificed at various ages (between 2 and 48 weeks old) and serum miRNAs analysed by RT-qPCR (Figure 3A–C). Pip6e-PMO treated mdx mice were administered with a single 12.5 mg/kg intravenous injection at 12 weeks of age and TA, diaphragm and heart muscles subsequently dissected and dystrophin restoration determined by quantitative immunofluorescence (Figure 3D) and RT-qPCR (Figure 3E) at various time points.Figure 3.


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)

Time course of serum dystromiR abundance. Male C57Bl/10, mdx and Pip6e-PMO-treated mdx mice were sacrificed at various ages, and serum miRNA levels were determined by small RNA TaqMan RT-qPCR. (A) miR-1, (B) miR-133a and (C) miR-206 abundance was normalized to an external spike-in control. All miRNA expression data were normalized to the mean of the 8-week-old C57Bl/10 group. Arrow indicates time of injection (single intravenous 12.5 mg/kg dose Pip6e-PMO). The time course of exon skipping and dystrophin restoration was also determined in the TA of the treated mdx samples at various time points by (D) quantitative immunofluorescence and (E) RT-qPCR, respectively. Values are mean ± SEM, n = 3–8.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt724-F3: Time course of serum dystromiR abundance. Male C57Bl/10, mdx and Pip6e-PMO-treated mdx mice were sacrificed at various ages, and serum miRNA levels were determined by small RNA TaqMan RT-qPCR. (A) miR-1, (B) miR-133a and (C) miR-206 abundance was normalized to an external spike-in control. All miRNA expression data were normalized to the mean of the 8-week-old C57Bl/10 group. Arrow indicates time of injection (single intravenous 12.5 mg/kg dose Pip6e-PMO). The time course of exon skipping and dystrophin restoration was also determined in the TA of the treated mdx samples at various time points by (D) quantitative immunofluorescence and (E) RT-qPCR, respectively. Values are mean ± SEM, n = 3–8.
Mentions: To investigate how serum miRNA levels change during disease progression or in response to dystrophin restoration C57Bl/10, mdx and mdx mice treated with Pip6e-PMO were sacrificed at various ages (between 2 and 48 weeks old) and serum miRNAs analysed by RT-qPCR (Figure 3A–C). Pip6e-PMO treated mdx mice were administered with a single 12.5 mg/kg intravenous injection at 12 weeks of age and TA, diaphragm and heart muscles subsequently dissected and dystrophin restoration determined by quantitative immunofluorescence (Figure 3D) and RT-qPCR (Figure 3E) at various time points.Figure 3.

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