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Amikacin Inhibits miR-497 Maturation and Exerts Post-ischemic Neuroprotection

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Abstract

MicroRNAs (miRNAs) are a group of small non-coding RNAs that regulate numerous signaling pathways involved in cerebral ischemia reperfusion injury. Recent finding demonstrated that miR-497 promotes ischemic neuronal death by negatively regulating anti-apoptotic proteins and therefore serves as a promising therapeutic target for cerebral ischemic injury. In this study, we present a systematic computational approach that includes 3D modeling, docking-based virtual screening, and molecular dynamics simulation to identify small-molecule inhibitors of pre-miR-497 maturation. The top hit, aminoglycosidic antibiotic, amikacin, formed a stable complex with pre-miR-497. Later, the protective efficacy of amikacin was evaluated against oxygen-glucose deprivation (OGD) and reoxygenation-induced neuronal cell death in SH-SY5Y cells and mouse organotypic hippocampal slice cultures. To confirm the inhibitory potential of amikacin on miR-497 maturation, quantitative real-time PCR was performed to check the expression of bcl-2, one of the primary anti-apoptotic targets of miR-497. Additionally, the expression level of mature miR-497 was quantified using TaqMan® MiRNA Assay Kit. Amikacin treatment effectively reduced OGD-induced cell death compared to control groups both in vitro and organotypic hippocampal slice cultures. Further, amikacin effectively increased the expression of bcl-2 in SH-SY5Y cells subjected to OGD. Interestingly, SH-SY5Y cells treated with amikacin displayed decreased expression of miR-497, probably due to inhibition of pre-miRic form. Our study provides strong evidence that amikacin inhibits miR-497 maturation and promotes ischemic neuronal survival by upregulating anti-apoptotic protein, bcl-2. Future studies directed at evaluating the neuroprotective efficacy and mechanism of amikacin animal models may lead to new therapeutic opportunities for preventing neuronal death after stroke.

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Acknowledgments

The computational portion of the study was funded by the Department of Biotechnology, Government of India “Bioinformatics Infrastructure Facility for Biology Teaching through Bioinformatics (BIF-BTBI)” (Grant number: BT/BI/25/001/2006 dated 25/03/2011). We would like to thank the Federal Commission for Scholarships for Foreign Students, Switzerland, for granting the visiting doctoral fellowship (Swiss Government Excellence Scholarship) (ESKAS-Nr: 2014.0803) to carry out research work at the University of Basel. The authors would like to thank Markus Saxer for the technical assistance.

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Authors and Affiliations

  1. School of Biotechnology, DBT - Centre for Bioinformatics, National Institute of Technology Calicut, Calicut, 673601, India

    S. Sinoy, S. M. Fayaz, K. D. Charles, V. K. Suvanish & G. K. Rajanikant

  2. Department of Biotechnology, Manipal Institute of Technology, Manipal University, Manipal, 576104, India

    S. M. Fayaz

  3. Anatomical Institute, Department of Biomedicine Basel, University of Basel, Pestalozzistrasse 20, 4056, Basel, Switzerland

    Josef P. Kapfhammer

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  1. S. Sinoy
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Correspondence to G. K. Rajanikant.

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All animal experiments were carried out at the University of Basel in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and permitted by the animal care committee of the Canton of Basel.

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The authors declare that they have no conflict of interest.

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Workflow of the methodology employed in the present study. (GIF 7293 kb)

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Sinoy, S., Fayaz, S.M., Charles, K.D. et al. Amikacin Inhibits miR-497 Maturation and Exerts Post-ischemic Neuroprotection. Mol Neurobiol 54, 3683–3694 (2017). https://doi.org/10.1007/s12035-016-9940-0

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  • DOI: https://doi.org/10.1007/s12035-016-9940-0

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