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Cellular and Molecular Neurobiology

, Volume 39, Issue 2, pp 223–240 | Cite as

miR-9 Upregulation Integrates Post-ischemic Neuronal Survival and Regeneration In Vitro

  • Sreekala S. Nampoothiri
  • G. K. RajanikantEmail author
Original Research
  • 106 Downloads

Abstract

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.

Keywords

MiRNA-9 HDAC4 Glucosamine Ischemic stroke Neuron regeneration Proliferation Neuroprotection Drug repurposing 

Abbreviations

ANOVA

Analysis of variance

ATP

Adenosine triphosphate

BCL2

B-cell lymphoma 2

BrdU

5-Bromo-2´-deoxyuridine

DARTS

Drug affinity responsive target stability

DMEM

Dulbecco’s modified Eagle’s medium

EBSS

Earle’s balanced salt solution

FBS

Fetal bovine serum

FDA

Food and drug administration

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

HDAC4

Histone deacetylase-4

IL-1β

Interleukin-1β

iNOS

Inducible nitric oxide synthase

LDH

Lactate dehydrogenase

MALDI-TOF MS

Matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry

MD

Molecular dynamics

MTT

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

NFκB1

Nuclear Factor Kappa B Subunit 1

OGD

Oxygen-glucose deprivation

PBS

Phosphate-buffered saline

PDB

Protein data bank

PI

Propidium iodide

qRT-PCR

Quantitative real time-polymerase chain reaction

RMSD

Root-mean-square deviation

RMSF

Root-mean-square fluctuation

SDS

Sodium dodecyl sulfate

SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SEM

Standard error of the mean

TNF-α

Tumor necrosis factor-α

VEGF

Vascular endothelial growth factor

Notes

Acknowledgements

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).

Author Contributions

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.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10571_2018_642_MOESM1_ESM.tif (585 kb)
Supplementary material 1 (TIF 584 KB)

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Authors and Affiliations

  1. 1.School of BiotechnologyNational Institute of Technology CalicutCalicutIndia

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