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Antisense oligonucleotides and short interfering RNAs silencing the cyclin-dependent kinase inhibitor p21 improve proliferation of Duchenne muscular dystrophy patients’ primary skeletal myoblasts

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Abstract

Increased levels of the cyclin-dependent kinase inhibitor p21 associated with decreased myoblast proliferation may be involved in the dystrophic process in Duchenne muscular dystrophy (DMD). Therefore we are interested to improve the proliferation of primary myoblasts of DMD patients by a reduction in p21 using either antisense oligonucleotides (ASO) or short interfering RNAs (siRNA). After transient transfection of myoblasts in cell culture proliferation was analyzed using a 5-bromo-2′-deoxyuridine assay comparing specific transfected cells with untransfected cells and cells transfected with scrambled ASO and luciferase siRNA, respectively. Four of five Dystrophin-deficient (Dys−) cell culture samples revealed an increase in proliferation between 7% and 18% compared to untransfected cells and between 8% and 36% compared to cells transfected with scrambled ASO. Transfection with siRNA was performed for selected samples to determine whether siRNA is more effective in gene silencing than ASO. The increase in proliferation using luciferase siRNA as reference was comparable to or less than ASO data using scrambled ASO as reference. Using untransfected cells as reference, the increase in proliferation was higher for siRNA than ASO (20–47% vs. 7–18%), but the data must be carefully interpreted with respect to nonspecific effects on gene expression by siRNA. Our findings of transient p21 gene silencing represent a basis for viral vector-mediated drug-inducible p21 shRNA expression in Dys− myoblasts which might enhance, prolong and regulate the proliferation effect.

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Abbreviations

ASO :

Antisense oligonucleotide

BMD :

Becker muscular dystrophy

BrdU :

5-Bromo-2′-deoxyuridine

DMD :

Duchenne muscular dystrophy

MTT :

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

siRNA :

Short interfering RNA

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Acknowledgements

The support of Marco Sifringer, Sabine Bucher, Boris Thurisch, and Stefanie Grunwald are gratefully acknowledged. We thank E. Schmidtmeyer for expert technical assistance. Human myoblast cultures were obtained from the Muscle Tissue Culture Collection at the Friedrich-Baur-Institute (Department of Neurology, Ludwig Maximilian University, Munich, Germany). The Muscle Tissue Culture Collection is part of the German network on muscular dystrophies (MD-NET, 01GM0302) funded by the German Ministry of Education and Research (BMBF, Bonn, Germany). The Muscle Tissue Culture Collection is a partner of Eurobiobank (Eurordis; scientific coordinator: Dr. C. Jaeger, Paris, France) funded by the EC within the 5th framework (QLRT-2001-02769). This research was supported in part by BMBF/AiF grant (1703402). S.E. is a fellow of the Hypatia Programm, University of Applied Sciences.

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Authors and Affiliations

  1. Department of Biotechnology, University of Applied Sciences, Seestrasse 64, 13347, Berlin, Germany

    Stefanie Endesfelder, Alexander Kliche & Astrid Speer

  2. Department of Neurology, Friedrich-Baur-Institut, and Gene Center, Klinikum der Universität-Innenstadt, Ludwig Maximilian University, Ziemsstrasse 1a, 80336, Munich, Germany

    Hanns Lochmüller

  3. Department of Neuropediatrics, Charité, Humboldt University Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

    Arpad von Moers

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  1. Stefanie Endesfelder
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  2. Alexander Kliche
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  3. Hanns Lochmüller
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  4. Arpad von Moers
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  5. Astrid Speer
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Correspondence to Astrid Speer.

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S. Endesfelder and A. Kliche contributed equally to this work

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Endesfelder, S., Kliche, A., Lochmüller, H. et al. Antisense oligonucleotides and short interfering RNAs silencing the cyclin-dependent kinase inhibitor p21 improve proliferation of Duchenne muscular dystrophy patients’ primary skeletal myoblasts. J Mol Med 83, 64–71 (2005). https://doi.org/10.1007/s00109-004-0607-3

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