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SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples

  1. Emanuela Abiusi
  2. Paola Infante
  3. Cinzia Cagnoli
  4. Ludovica Lospinoso Severini
  5. Marika Pane
  6. Giorgia Coratti
  7. Maria Carmela Pera
  8. Adele D'Amico
  9. Federica Diano
  10. Agnese Novelli
  11. Serena Spartano
  12. Stefania Fiori
  13. Giovanni Baranello
  14. Isabella Moroni
  15. Marina Mora
  16. Maria Barbara Pasanisi
  17. Krizia Pocino
  18. Loredana Le Pera
  19. Davide D'Amico
  20. Lorena Travaglini
  21. Francesco Ria
  22. Claudio Bruno
  23. Denise Locatelli
  24. Enrico Silvio Bertini
  25. Lucia Ovidia Morandi
  26. Eugenio Mercuri
  27. Lucia Di Marcotullio
  28. Francesco Danilo Tiziano  Is a corresponding author
  1. Department of Life Sciences and Public Health, Section of Genomic Medicine, Università cattolica del Sacro Cuore, Italy
  2. Center For Life Nano Science@Sapienza, Istituto Italiano di Tecnologia; Department of Molecular Medicine, Università degli Studi di Roma "La Sapienza", Roma, Italy, Italy
  3. Clinical and Experimental Epileptology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy, Italy
  4. Department of Molecular Medicine, Università degli Studi di Roma "La Sapienza", Italy
  5. Pediatric Neurology, Department of Woman and Child Health and Public Health, Fondazione Policlinico Universitario A. Gemelli IRCCS-Università Cattolica del Sacro Cuore, Italy
  6. Centro Clinico Nemo, Fondazione Policlinico Universitario A. Gemelli IRCCS-Università Cattolica del Sacro Cuore, Italy
  7. Unit of Neuromuscular and Neurodegenerative Disorders, Dept. Neurosciences, Bambino Gesu' Children's Hospital IRCCS, Italy
  8. Department of Pediatric Neuroscience, Fondazione IRCCS Istituto Neurologico Carlo Besta, Italy
  9. Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Italy
  10. Department of Medical and Surgical Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Italy
  11. Bioenergetics and Molecular Biotechnologies (IBIOM), CNR-Institute of Biomembranes, Italy
  12. CNR-Institute of Molecular Biology and Pathology (IBPM), Italy
  13. Amazentis SA, EPFL Innovation Park, Switzerland
  14. Department of Translational Medicine and Surgery, Section of General Pathology, Università Cattolica del Sacro Cuore, Italy
  15. Fondazione Policlinico Universitario A. Gemelli - IRCCS, Italy
  16. Center of Translational and Experimental Myology, IRCCS Istituto Giannina Gaslini, Italy
  17. Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University, Italy
  18. Unit of Medical Genetics, Department of Laboratory science and Infectious Diseases, Fondazione Policlinico Universitario IRCCS “A. Gemelli”, Italy
Research Article
Cite this article as: eLife 2021;10:e68054 doi: 10.7554/eLife.68054
5 figures, 2 tables and 6 additional files

Figures

Heatmaps obtained by the whole miRNome analysis of muscle biopsies (A), myoblasts (B), and myotubes (C) of spinal muscular atrophy (SMA) patients and controls; patient and control samples display a separate clusterization.

99, 20, and 19 miRs were found deregulated in SMA in muscle biopsies, myoblasts, and myotubes, respectively; (D) Venn’s diagram showing the five miRNAs shared among the three groups, three between myoblasts and myotubes, two between myoblasts and biopsies, and three between myotubes and biopsies.

Validation pipeline of miRNAs identified by whole miRNome analysis in serum samples of patients and controls.

‘Others’ indicates miRs that were identified in other studies or with key function in skeletal muscle.

Figure 3 with 4 supplements
The SMA-miRs (miR-181a-5p [A], miR-324-5p [B] and miR-451a [C]) were significantly upregulated in serum samples of spinal muscular atrophy (SMA) patients (p=4.3 * 10–4; 0.02; 0.004, respectively).

Receiver operating characteristic (ROC) curves showed that the quantification of SMA-miRs has 80% sensitivity and 75% specificity in distinguishing patients from controls (D). Correlation of miR-324-5p with SMA type (E): the levels in SMA II and SMA III patients were significantly increased compared to those of SMA I patients (p=0.03 and 0.04, respectively).

Figure 3—figure supplement 1
Multiple variable correlation of miR-181a-5p, -324-5p, and -415a levels and age at sampling.

In red, the only significant correlation, between miR-181a-5p and miR-451a (p=0.0002).

Figure 3—figure supplement 2
Comparison of levels of miR-181a-5p, -324-5p, and -451a in male and female patients; only miR-181a-5p showed a significant difference in females compared to males (*p=0.024).
Figure 3—figure supplement 3
Analysis of type II and III patients with three SMN2 copies; the two groups were not different for miRs levels (p>0.05, A) but showed a significant difference in age (**p=0.0092, B).
Figure 3—figure supplement 4
Transfections of SH-SY5Y neuroblastoma cells with SMA-miR mimics (final concentration: 50 or 100 nM).

In spite of the huge increase in SMA-miR levels (A), SMN1/SMN2 transcripts remained unchanged, except for the SMNΔ7 isoform in cells treated with miR-324-5p, which was reduced by 50%, independently of the mimic concentration (B).

Figure 4 with 1 supplement
Survival curves of SMNΔ7-mice treated with intrathecal injection of anti-miR-181a-5p (n = 36) and untreated (n = 71); the overall survival remained unchanged (p>0.05).
Figure 4—figure supplement 1
SMNΔ7 mice treated with anti-miR-324-5p showed a significant transient increase in body weight, between P7 and P10 (p=0.002).
Figure 5 with 1 supplement
The spinal muscular atrophy score (SMA-score) predicts the phenotypic severity in SMA patients.

Correlation between the SMA-score and the clinical decimal SMA subtype in the whole cohort (A) and aged <6 years (B). Red circles are individual samples, the blue line indicates the expected distribution, the green line indicates the 95% confidence interval, and the black lines are the prediction interval.

Figure 5—figure supplement 1
Linear correlation analysis among SMN2 copy number and spinal muscular atrophy (SMA) type, estimated by the standard classification (A; R2 = 52.45%, n = 41, p<10–5) and the decimal classification (B; R2 = 67.04%, n = 39, p<10-5).

Correlation with SMA-score and SMA type estimated by the decimal classification in patients with three SMN2 copies (C; R2 = 30.04, n = 21, p=0.008). Linear correlation analysis among SMA-scores obtained with the two equations (all ages vs. <6 years) (D; R2 = 80.31, n = 21, p<10-5). Red circles are individual samples, the blue line indicates the expected distribution, the green line indicate the 95% confidence interval, and the black lines are the prediction interval.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)SMN1GenBankHGNC:HGNC:11,117
Gene (Homo sapiens)SMN2GenBankHGNC:HGNC:11,118
miR (Homo sapiens)hsa-miR-181a-5pmiRBaseMIMAT0000256
miR (Homo sapiens)hsa-miR-324-5pmiRBaseMIMAT0000761
miR (Homo sapiens)hsa-miR-451amiRBaseMIMAT0001631
Strain, strain background (Mus musculus)SMNΔ7 miceFVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4,299Ahmb/JJackson LaboratoryStock number: 005025Hum Mol Genet 14(6):845–57, 2005
Genetic reagent (Homo sapiens)miRCURY LNA microRNA mimic: hsa-miR-181a-5p, hsa-miR-324-5p, hsa-miR-451aExiqon50–100–200 nM
Genetic reagent (Homo sapiens)miRCURY LNA microRNA antogomiR: hsa-miR-181a-5p, hsa-miR-324-5p, hsa-miR-451aExiqon0.1 nM
Cell line (Homo sapiens)Primary myoblastsItalian Telethon Network of Genetic Biobanks6756, 6760, 6762,
6816, 7147, 8823,
8655, 8537
Biological sample (Homo sapiens)Muscular biopsiesItalian Telethon Network of Genetic Biobanks10370, 10351, 8023,
4688, 10543, 10583,
7669, 5944, 5824,
5717, 6760, 6438,
6082, 5379, 7689,
5842, 9814
Sequence-based reagentSee Supplementary file 3IDT (Integrated DNA Technologies)
Commercial assay or kitTruSeq Small RNA Sample Preparation kitIlluminaTruSeq Small RNA
Library Prep Kit –
RS-200-0024
Commercial assay or kitmiRCURY RNA Isolation Kit – BiofluidsExiqon
Commercial assay or kitUniversal cDNA synthesis kit IIExiqon
Commercial assay or kitPick-&-Mix miRNA PCR Panel 96-wellExiqon
Commercial assay or kitE.Z.N.A PX Blood RNA KitOmega bio-tekSKU: R1057-01
Commercial assay or kitHigh Capacity cDNA Reverse Transcription KitThermo Fisher ScientificCatalog number: 4368814
Software, algorithmIllumina Genome AnalyzerIllumina
Software, algorithmRealTime StatMinerVersion 4.1
Software, algorithmStatgraphics Centurion XV softwareStatPoint Inc
Software, algorithmSPSS 18.0 softwareSPSSRRID:SCR_002865
Table 1
miRs differentially expressed in spinal muscular atrophy (SMA), as reported in previous studies.
hsa-miRmiRNomeRelative qPCRAbsolute qPCR*Reference
miR-19a-3pUpregulatedUpregulated<10 molecules/µl serum; p=0.42Haramati et al., 2010; Gonçalves et al., 2018
miR-23a-3pDownregulatedUpregulated150–200 molecules/µl serum; 10 patients/controls analyzed; p=0.62Kaifer et al., 2019
miR-206NonsignificantUpregulated50–100 molecules/µl serum; 15 patients/controls analyzed; p=0.24Valsecchi et al., 2015; Catapano et al., 2016; Bonanno et al., 2020
miR-9NonsignificantNot tested<10 molecules/µl serum; p=0.30Catapano et al., 2016
miR-132NonsignificantNot testedNot testedCatapano et al., 2016
miR-146aUpregulatedUpregulated<5 molecules/µl serum; p=0.10Sison et al., 2017
4miR-431NonsignificantNot testedNot testedWertz et al., 2016
miR-183NonsignificantNot testedNot testedKye et al., 2014
  1. *

    p-Values refer to the significance of comparison of the miR levels in patients and controls by Mann–Whitney U-test. p-values < 0.05 were considered significant.

Additional files

Supplementary file 1

Demographic and genetic characteristics of subjects who underwent muscle biopsy.

https://cdn.elifesciences.org/articles/68054/elife-68054-supp1-v3.docx
Supplementary file 2

Clinical and molecular characteristics of subjects included in the present study.

https://cdn.elifesciences.org/articles/68054/elife-68054-supp2-v3.docx
Supplementary file 3

Primer sequences.

https://cdn.elifesciences.org/articles/68054/elife-68054-supp3-v3.docx
Supplementary file 4

List of deregulated miRs in whole miRNome analyses (patients vs. controls).

https://cdn.elifesciences.org/articles/68054/elife-68054-supp4-v3.docx
Supplementary file 5

Assessment by relative and/or absolute qPCR of miR levels in serum samples of patients and controls.

https://cdn.elifesciences.org/articles/68054/elife-68054-supp5-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/68054/elife-68054-transrepform1-v3.docx

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