Elsevier

Experimental Neurology

Volume 286, December 2016, Pages 137-146
Experimental Neurology

Research Paper
Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

https://doi.org/10.1016/j.expneurol.2016.10.003Get rights and content

Highlights

  • Pre-miR-21 markedly increases following status epilepticus (SE).

  • Pre-miR-21 localizes to polysomes with translating mRNAs.

  • Specific binding competition between pre- and mature miR-21 for 3’UTR of TGFBR2 mRNA de-represses miR-21-mediated gene suppression in vitro.

  • TGFBR2 increases and NT-3 decreases following SE, implicating pre-miR-21-mediated gene regulation.

Abstract

MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine-induced status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3′UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-β receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene.

Introduction

MicroRNAs (miRNAs) are short non-coding RNA molecules (~ 20–25 nucleotides) that regulate gene expression of target messenger RNAs (mRNAs) post-transcriptionally in diverse cellular pathways (Kim, 2005). MiRNAs bind to mRNAs blocking translation and/or enhancing degradation, allowing for a large number of proteins to be concurrently regulated. To date, there are hundreds of miRNA members identified in higher eukaryotes targeting > 60% of protein-encoding genes (Bartel, 2004, Friedman et al., 2009b, Kim, 2005). In miRNA biogenesis, primary miRNAs (pri-miRNAs) are first transcribed by RNA polymerase II and cleaved into precursor miRNAs (pre-miRNAs) by the RNase III Drosha in the nucleus; pre-miRNAs are then exported into the cytoplasm where they are processed by another RNase III Dicer to produce a short miRNA duplex consisting of a guide strand (mature miRNA) and passenger strand (miRNA*). The mature miRNA and Argonaute (Ago2) form a RNA-induced silencing complex to target and silence mRNAs, whereas the miRNA* is often rapidly degraded (Ha and Kim, 2014, Kosik, 2006). The miRNA intermediates were merely considered as by-products with no biological function until recent in vitro studies. Trujillo et al. described function of pri-miRNAs in target recognition and repression while Okamura et al. showed miRNA-type repression of specific miRNA stem loops (Okamura et al., 2013, Trujillo et al., 2010). Our previous study demonstrated competition between pre- and mature miRNAs for binding to the 3′UTR of their target mRNAs (Roy-Chaudhuri et al., 2014). However, the physiological consequences of these additional layers of translational regulation by miRNA intermediates have not been explored in epilepsy or other disease models.

Alterations in miRNA expression have been implicated in a wide variety of neurological disorders, including epilepsy (Bian and Sun, 2011, Bot et al., 2013, Jimenez-Mateos and Henshall, 2013, Kosik, 2006). Epilepsy is one of the most common diseases of the nervous system with a lifetime incidence of 3% and is associated with comorbid learning and memory problems (de Boer et al., 2008, Hesdorffer et al., 2011, Jimenez-Mateos et al., 2012, Pohlmann-Eden et al., 2006). Development of epilepsy after a neurologic insult has been associated with a large number of cellular and molecular changes, such as neuronal loss, gliosis, neurogenesis, synaptic organization, and alternation of gene expression including miRNAs (Pitkanen and Lukasiuk, 2009). Indeed, miRNA dysregulation is observed in a variety of epilepsy models and in tissue from patients undergoing surgery for medically refractory epilepsy (Dogini et al., 2013, Henshall, 2014). Altered miRNA regulation has been implicated in apoptosis, neurogenesis, synaptic functions, inflammation, and gliosis (Dogini et al., 2013, Jimenez-Mateos and Henshall, 2013). However, further studies are needed to understand how miRNAs regulate epileptogenesis and if they provide a novel therapy to treat epilepsy.

Studies on epileptogenesis use models that have high rates of epilepsy following an initial insult. Treating rodents with pilocarpine, a chemoconvulsant, is a widely used model to induce status epilepticus (SE, a prolonged seizure) from which the animals initially recover but go on to develop spontaneous seizures or epilepsy (Curia et al., 2008). Animals present behavioral, electrographic, and neuropathologic features in the limbic structures similar to those observed in patients with temporal lobe epilepsy. MicroRNA-21 (miR-21), an evolutionarily conserved miRNA, increases following a variety of brain insults including head trauma, stroke, and SE that are associated with increased risk of developing epilepsy (Buller et al., 2010, Gorter et al., 2014, Peng et al., 2013).

Here, we observed differential expression of miR-21 precursors and mature miR-21 in the hippocampus following pilocarpine SE. To understand the functional significance of differential levels of precursor and mature miR-21 following SE, we carried out bioinformatics and biochemical approaches to show that pre-miR-21 can compete with mature miR-21 for regulating mRNA levels of Transforming Growth Factor β Receptor 2 (TGFBR2), but not Neurotrophin-3 (NT-3), both of which are targets of mature miR-21. TGF-β is a master anti-inflammation cytokine controlling neurological and immune functions following a neurologic insult and binds to TGF-β receptor 1 and receptor 2 (TGFBR1 and TGFBR2, respectively) resulting in a cascade of molecular changes implicated in epileptogenesis (Cacheaux et al., 2009, Friedman et al., 2009a, Okamoto et al., 2010). Together, our data suggest that pre-miR-21 may compete with mature miR-21 and block miRNA-mediated mRNA degradation and/or translational repression in the hippocampus following SE. This possibly contributes to the prolonged activation of TGF-β signaling with implications for epileptogenesis.

Section snippets

Differential changes in expression of pri-miR-21, pre-miR-21, and mature miR-21 in the hippocampus following status epilepticus

Pri-miR-21, pre-miR-21 and mature miR-21 levels in the rat hippocampi were assayed using qRT-PCR and northern blot analysis 4, 12, and 48 h after the animals had experienced a pilocarpine-induced Racine stage V seizure (Racine, 1972) (Supplementary Fig. 1). The SE animals exhibited substantial increases in each transcript compared to controls, but each transcript displayed a unique time course (Fig. 1). The qRT-PCR data showed that pri-miR-21 levels did not increase at 4 h post SE but increased

Discussion

To our knowledge, this is the first study focusing on precursor miRNA expression and its novel regulation of miRNA activity in status epilepticus. In our prior study we showed that complementary sequences exist for both the miR-151-5p and -3p sequences within the 3′UTR of the transcription factor E2f6 gene (Roy-Chaudhuri et al., 2014). Pre-miR-151 binds to and competes with mature miR-151 (miR-151-5p) binding to the overlapping 3′UTR region of E2f6. In contrast, only mature miR-151 is capable

Animals and induction of status epilepticus

Adult male Sprague Dawley rats (Charles River Laboratories International, CA) between 60 and 90 days of age were provided with unrestricted access to food and water in temperature and humidity controlled housing with a 12 h light–12 h dark cycle (lights on 7:00 AM). The rats were injected with 1 mg/kg methyl-scopolamine (Sigma-Aldrich, MO) intraperitoneally (IP) to block peripheral cholinergic effects 30 min prior to pilocarpine-induced status epilepticus (SE). 385 mg/kg pilocarpine hydrochloride

Acknowledgements

We thank Deepti Dueby for review of this manuscript. This work was supported by National Institute of Neurological Disorders and Stroke (NINDS) (NSRO1 N5056222) (B.E.P) and RO1 DK078424 (M.A.K.). K.C. is supported by the Child Health Research Institute, Lucile Packard Foundation for Children's Health and the Stanford CTSA (UL1 TR001085) from Stanford University.

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