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

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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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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Fig. S1
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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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Keywords

  • miRNA inhibition
  • miRNA modeling
  • Virtual screening
  • miRNA MD simulation