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

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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.

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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

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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).

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  1. School of Biotechnology, National Institute of Technology Calicut, Calicut, 673601, India

    Sreekala S. Nampoothiri & G. K. Rajanikant

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  1. Sreekala S. Nampoothiri
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  2. G. K. Rajanikant
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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.

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Correspondence to G. K. Rajanikant.

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Nampoothiri, S.S., Rajanikant, G.K. miR-9 Upregulation Integrates Post-ischemic Neuronal Survival and Regeneration In Vitro. Cell Mol Neurobiol 39, 223–240 (2019). https://doi.org/10.1007/s10571-018-0642-1

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