Temporal Differences in MicroRNA Expression Patterns in Astrocytes and Neurons after Ischemic Injury

Alexander J Annala,Mateo Ziu,Lauren Fletcher,Murat Digicaylioglu,Shushan Rana,David F Jimenez

doi:10.1371/journal.pone.0014724

Alexander J Annala, Mateo Ziu + Show 4 more

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https://doi.org/10.1371/journal.pone.0014724

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Abstract

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions.

Highlights

The discovery of microRNAs, a novel class of small non-protein-coding molecules, has introduced a whole new level and mechanism of gene regulation[1,2,3,4,5]
We show that a known neuroprotectant, Insulin-Like Growth Factor I (IGF-I), can reduce the expression of miR-29b, which is highly upregulated in ischemic neurons
We examined temporal changes in expression levels of a specific set of miRNAs in neurons and astrocytes at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after the oxygen-glucose deprivation (OGD) as compared to cells growing in normal conditions

Summary

Introduction

The discovery of microRNAs (miRNAs), a novel class of small non-protein-coding molecules, has introduced a whole new level and mechanism of gene regulation[1,2,3,4,5]. MiRNAs regulate gene expression at the post-transcriptional level by altering translation of target messenger RNAs (mRNAs) into proteins. The mechanisms by which miRNAs function is by inhibition of the protein translation or by promotion of mRNA decay[7]. By targeting the mRNA of protein-coding genes, miRNAs play a critical role in development, control of cell growth, proliferation, metabolism and in a variety of other biological processes[6]. MiRNAs have been implicated as well in the etiology of a variety of pathological processes such as cancer, ischemia, developmental arrest, inflammatory diseases and cellular response to stressful conditions [7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37]

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