miR-9 Upregulation Integrates Post-ischemic Neuronal Survival and Regeneration In Vitro
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The irrefutable change in the expression of brain-enriched microRNAs (miRNAs) following ischemic stroke has promoted the development of radical miRNA-based therapeutics encompassing neuroprotection and neuronal restoration. Our previous report on the systems-level prediction of miR-9 in post-stroke-induced neurogenesis served as a premise to experimentally uncover the functional role of miR-9 in post-ischemic neuronal survival and regeneration. The oxygen-glucose deprivation (OGD) in SH-SY5Y cells significantly reduced miR-9 expression, while miR-9 mimic transfection enhanced post-ischemic neuronal cell viability. The next major objective involved the execution of a drug repositioning strategy to augment miR-9 expression via structure-based screening of Food and Drug Administration (FDA)-approved drugs that bind to Histone Deacetylase 4 (HDAC4), a known miR-9 target. Glucosamine emerged as the top hit and its binding potential to HDAC4 was verified by Molecular Dynamics (MD) Simulation, Drug Affinity Responsive Target Stability (DARTS) assay, and MALDI-TOF MS. It was intriguing that the glucosamine treatment 1-h post-OGD was associated with the increased miR-9 level as well as enhanced neuronal viability. miR-9 mimic or post-OGD glucosamine treatment significantly increased the cellular proliferation (BrdU assay), while the neurite outgrowth assay displayed elongated neurites. The enhanced BCL2 and VEGF parallel with the reduced NFκB1, TNF-α, IL-1β, and iNOS mRNA levels in miR-9 mimic or glucosamine-treated cells further substantiated their post-ischemic neuroprotective and regenerative efficacy. Hence, this study unleashes a potential therapeutic approach that integrates neuronal survival and regeneration via small-molecule-based regulation of miR-9 favoring long-term recovery against ischemic stroke.
KeywordsMiRNA-9 HDAC4 Glucosamine Ischemic stroke Neuron regeneration Proliferation Neuroprotection Drug repurposing
Analysis of variance
B-cell lymphoma 2
Drug affinity responsive target stability
Dulbecco’s modified Eagle’s medium
Earle’s balanced salt solution
Fetal bovine serum
Food and drug administration
Glyceraldehyde 3-phosphate dehydrogenase
Inducible nitric oxide synthase
- MALDI-TOF MS
Matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry
Nuclear Factor Kappa B Subunit 1
Protein data bank
Quantitative real time-polymerase chain reaction
Sodium dodecyl sulfate
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Standard error of the mean
Tumor necrosis factor-α
Vascular endothelial growth factor
This study was funded by (a) the Department of Biotechnology, Government of India “Bioinformatics Infrastructure Facility for Biology Teaching through Bioinformatics (BIFBTBI)” (Grant Number: BT/BI/25/001/2006 dated 25/03/2011) and (b) Kerala State Council for Science, Technology and Environment, Science Research Scheme (Grant Number: 018/SRSLS/2014/CSTE).
SSN and RGK designed experiments; SSN performed experiments, analyzed data, wrote the manuscript; RGK revised the manuscript critically and approved the final version to be submitted.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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