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. 2019 Jun 26;71(4):809–818. doi: 10.1007/s10616-019-00324-3

MiR-206 inhibits epilepsy and seizure-induced brain injury by targeting CCL2

Zhenggang Wu 1,✉, Ying Liu 1, Jing Huang 1, Yujing Huang 1, Lin Fan 1
PMCID: PMC6663963  PMID: 31243650

Abstract

To determine the function of miR-206 in epilepsy. Epileptic rat model was established by intra-amygdala injection of kainic acid (KA). Expression levels of miR-206, C–C Motif Chemokine Ligand 2 (CCL2) and interleukin-1β (Il-1β) in hippocampus tissues was measured by reverse transcription-quantitative PCR (RT-qPCR) and western blot. Dual luciferase reporter assay was performed to determine the binding of miR-206 to 3′ untranslated region (UTR) of CCL2. Finally, brain waves were recorded and Hematoxylin and eosin (HE) staining and Nissl’s staining were performed on the epileptic rat injected with LPS, miR-206 agomir, adeno-associated virus (AAV) expressed CCL2 alone or in combination. Expression of miR-206 was specially decreased in hippocampus tissues compared to cortex in response to KA induced pathologic brain activity. Enforced expression of miR-206 by injection miR-206 agomir not only decreased seizure activity, but also protected KA-induced neuronal loss. And enforced expression of miR-206 suppressed increase of C–C Motif Chemokine Ligand 2 (CCL2) and interleukin-1β (Il-1β) which were induced by injection of KA or KA combined with lipopolysaccharide (LPS). Further more, results of dual luciferase reporter assay confirmed CCL2 was a target of miR-206. Finally, co-injection adeno-associated virus (AAV) expressed CCL2 with miR-206 agomir abolished the function of miR-206 agomir. Taken together, our results showed that expression of miR-206 could inhibit seizure-induced brain injury by targeting CCL2. Our results showed that expression of miR-206 could inhibit seizure-induced brain injury by targeting CCL2.

Keywords: Epilepsy, miR-206, Hippocampus, CCL2, Inflammation

Introduction

Epilepsy, as one of the most common neurological disorder, affects around 3% of the population worldwide, and the overall prevalence of lifetime epilepsy in China steadily increased from 1.99‰ (95% CI 1.31–3.02) in 1990 to 7.15‰ (95% CI 3.98–12.82) in 2015 (Song et al. 2017). Epilepsy is predominantly characterized by recurring and unprovoked seizures which result from an imbalance in excitatory and inhibitory signal transmission of neurons in cerebral origin (Ke et al. 2017; Wahab et al. 2017). Although majority of epilepsy patients have good prognosis, up to 30% patients are refractory to medical treatment and the progress underlying recurrent seizures are not well understood (Wang et al. 2015). Thus, it is necessary to further explore the mechanism of epilepsy and develop new therapy strategy.

Increasing evidence supports that inflammatory pathways contribute to the pathogenesis of epilepsy (Lourdes Lorigados et al. 2013). Overexpression of chemokines and cytokines were widely identified in epilepsy. In pilocarpine-induced seizures, expressions of C–C Motif Chemokine Ligand 2 (CCL2), C–C Motif Chemokine Ligand 3 (CCL3), C–C Motif Chemokine Ligand 5 (CCL5), and interleukin-1β (IL-1β) were up regulated in piriform cortex, hippocampus, and neocortex of adult rats (Arisi et al. 2015). And inhibition biosynthesis of IL-1β reduced acute seizures and drug resistant chronic epileptic activity in mice (Maroso et al. 2011). These results suggest that inflammatory response plays important role in epilepsy.

microRNAs (miRNAs) are about 22 bp length non-coding RNAs that down-regulated expression levels of their targets through degradation of mRNA or inhibition of protein translation by directly binding to 3′ untranslated region (UTR) of their targets (Haenisch et al. 2015). Recent studies have showed that they might deeply involve in epilepsy. Firstly, detection of expression of miRNAs could be a promising tools in diagnosis for the epilepsy patients (Ma 2018). It has been found that expression levels of miR-181a changed dramatically in different stage of epilepsy (Ren et al. 2016). Besides, differentially expression of miRNAs in serum, such as miR-301a-3p, can also function as biomarkers for drug-resistant of epilepsy (Wang et al. 2015). Moreover, miRNAs could regulate neuronal excitability through various signaling. Expression of the brain-specific microRNA, miR-124, could inhibit neuronal firing and suppresses seizure activity mainly by targeting cAMP-response element-binding protein1 (CREB1) (Wang et al. 2016). In contrast, depletion of miR-134 prolonged seizure suppressant and neuroprotective actions (Jimenez-Mateos et al. 2012). Thus, the function of individual miRNA need further study.

In this study, we focused on miR-206. Functions of miR-206 in Alzheimer disease and schizophrenia indicated that miR-206 played an important role in neurological function (Hauberg et al. 2016; Lee et al. 2012; Xu et al. 2010). Further study using microRNA microarray showed that miR-206 differently expressed in drug resistant epilepsy mice model (Moon et al. 2014). These results indicated that miR-206 involved in epilepsy. However, the role of miR-206 hasn’t been studied. Thus, we tried to determine the function of miR-206 in epilepsy in this study.

Materials and methods

Animals and surgery

The experiments were conducted in accordance with the on Animal Care Committee of Taizhou People’s Hospital. Adult, male Sprague–Dawley rats (260–280 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Cat No.: 101, Beijing, China) and maintained in a 12–12-h light–dark cycle room with food water ad libitumat 25 °C.

The methods of kainic acid (KA) induced epilepsy rat model were modified from procedures reported previously (Liao et al. 2017). In brief, each rat was first anaesthetized via isoflurane (1–3%). Then, the head was fixed into a stereotaxic apparatus and prepared for aseptic surgery. A burr hole was drilled in the dorsal surface of the skull at the stereotaxic coordinates which was from bregma: − 0.2 mm anteroposterior (AP), + 1.0 mm mediolateral (ML). A 5 μl Hamilton syringe filled with KA solution (40 μg/kg in saline, K0250-50MG, Sigma-Aldrich, Germany) was placed over the burr, and the 26 gauge needle was lowered into the lateral ventricle (2 mm below endocranium). The injection rate was 0.2 µl/min, and the needle was left in place for 5 min and withdrawn slowly afterwards. For miRNA co-injection, 10 nmol/kg miR-206 agomir (scrambled control) was added in KA solution. For AAV injection, another injection was performed 6 h after the KA injection.

The methods of electroencephalography (EEG) recording were modified from procedures reported previously (Liao et al. 2017). Two polyamide insulated stainless steel recording electrode (diam = 0.1 mm, Plastics One Inc. Roanoke, VA, USA) were implanted bilateral at the skull above the hippocampus (AP − 1.5 mm and ML 1.8 mm) and a reference electrode were placed over the cerebellum. Seizure behaviors and EEG performance were recorded within 1 h after injection. The EEG signals were analyzed using Nicolet 1.0 (Nicolet Instruments, Madison, WI, USA). The behavioral manifestations of seizures were classified according to a modification of Racine’s classification (Racine 1972).

Primary hippocampal neuronsculture and treatment

The method for preparation of primary hippocampal neurons was according to the previous study (Sun et al. 2015). In brief, hippocampi from neonatal SD rats were dissected and rinsed in ice cold dissection buffer. After removed blood vessels and white matter, hippocampi were incubated with 0.125% trypsin in Hanks’ balanced salt solution (HBSS) at 37 °C for 20 min and then filtered through 200 mesh stainless steel. The filtered cell suspension was centrifuged and the pellet was re-suspended and cultured in DMEM/F-12 with 20% FBS, 100 U/l penicillin, 100 mg/l streptomycin and 0.5 mM glutamine. After 72 h, arabinosylcytosin (10 mg/l) was added to prevent the growth of non-neuronal cells.

miRNAs, plasimds and AAV

mimics of miR-206 and agomir of miR-206 and their negative control (NC) were purchased from GenePharmaCo.Ltd. (Shanghai, China). Wild type (WT) and mutants of 3′UTR of CCL2 were synthesized into pmirGLO (Promega, USA) vector by Genewiz Inc. (Suzhou, Jiangsu, China). Adeno-associated virus containing CCL2 was packed by Hanbio Co. Ltd. (Shanghai, China).

Luciferase assay

pmirGLO reporter plasmids containing WT or mutants of 3′UTR of CCL2 and miR-206 mimics or scramble control were co-delivered into 293t cells by Lipofectamine 3000 (L3000015, Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, cells were collected and lysed. Then, the firefly and renilla luciferase activities were determined using Dual-Luciferase® Reporter Assay System (E1910, Promega, USA) according to the manufacturer’s protocol.

Hematoxylin and eosin (HE) staining and Nissl’s staining

The animals were deeply anesthetized with chloral hydrate and transcardiac perfused with saline followed by ice-cold 4% phosphate-buffered paraformaldehyde. Brains were removed and immersion in 4% paraformaldehyde overnight. Then, coronal blocks were dehydrated with gradient ethanol and dimethyl benzene and embedded in paraffin. Sections were prepared for HE and Nissl’s staining.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) from tissues or cultured cells. Concentration of total RNA was measured using NanoDrop 2000 (NanoDrop; Thermo Fisher Scientific, Inc.). Reverse transcription was performed with PrimeScript™ RT reagent Kit (Takara Biotechnology Co., Ltd.) according to the manufacturer’s protocol. For miR-206, special primer sets were used as in perviously study (Zhou et al. 2013). RT-qPCR was performed with SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) on a Thermal Cycler Dice™ Real Time System III (Takara Biotechnology Co., Ltd.). The relative mRNA expression levels were calculated as 2−∆∆Ct.

Western blots

Tissues or collected cells were lysed in RIPA buffer (89900, Thermo Fisher Scientific, Inc.), and total protein concentration was determined using Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Inc.). Protein samples (20 μg) were loaded, separated by SDS-PAGE and transferred to PVDF membranes. Blots were incubated with the following primary antibodies: Anti-CCL2 (1:500; Cat. No. NBP1-07034; Novus Biologicals), anti-IL-1β (1:500; Cat. No. NBP1-42767; Novus Biologicals) and anti-β-actin (1:5000; Cat. No. ab8226; Abcam) at 4 °C overnight. After washing, blots were incubated with appropriate HRP-conjugated secondary antibodies for 45 min at room temperature. The blots were then developed with Pierce™ ECL Plus Western Blotting Substrate (32132, Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Densitometry of the blots were analyzed by ImageJ (ImageJ bundled with 64-bit Java 1.8.0_112, National Institutes of Health, USA).

Statistical analysis

Data was presented as mean ± SEM. Statistical analysis was performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). Differences between groups were evaluated using Student’s t test for two groups or Tukey’s multiple comparisons test after ANOVA test for three or more groups. All Significance level was defined as P value < 0.05 and abbreviated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Results

miR-206 was down regulated in hippocampus in response to KA

In order to find out the function of miR-206 in epilepsy, we first determined whether pathologic brain activity affects expression levels of miR-206. KA was injected into right lateral ventricle of rats to induce status epilepticus (Hellier et al. 1998), and the brainwaves recorded by electroencephalograph (EEG) from KA administrated group were spectral compared with control group (Fig. 1a). Both the amplitude and spike frequency were much higher in KA administrated group than control group which indicated that pathologic brain activity was successfully induced by KA (Fig. 1a). Based on this, we determined expression levels of miR-206 in hippocampus compared with cortex. The results showed that expression of miR-206 was specially and obviously decreased in hippocampus in KA administrated group, whereas no obviously change in cortex was observed (Fig. 1b). Then, we isolated hippocampal neurons and determined the expression levels of miR-206 in hippocampal neurons in response to treatment of KA. The results showed that expression of miR-206 in hippocampal neurons treated with KA was continuously decreased in 24 h and also inversely proportional to the concentration of KA (Fig. 1c, d). These results suggested down-regulated miR-206 might involve in KA induced epilepsy.

Fig. 1.

Fig. 1

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Expression of miR-206 was downregulated in hippocampus in response to KA. a Amplitude and spike frequency of brain waves were upregulated in rats injected with KA. b Expression of miR-206 was specifically decreased in hippocampus. c Expression of miR-206 was decreased in the primary cultured hippocampal neurons treated with KA. d Expression of miR-206 was decreased in a does-dependent manner in the primary cultured hippocampal neurons treated with KA

Expression of miR-206 protected neurons from KA induced epilepsy

Since expression levels of miR-206 was specially decreased in hippocampus in response to KA (Fig. 1), we tried to determine the role of miR-206 in KA induced epilepsy by co-injection of miR-206 agomir with KA. We first determined the expression levels of miR-206 to validate the efficiency of miR-206 agomir. Expression levels of miR-206 were indeed decreased in KA group or KA + NC agomir group compared with control group, whereas co-injection of miR-206 agomir reversed the decreasing of miR-206 by KA (Fig. 2a). Then, we compared the brainwaves recorded by EEG. The amplitude and spike frequency of brainwaves in the group co-injected miR-206 agomir with KA was similar with the control group and obviously lower than KA group or KA + NC agomir group (Fig. 2b). Finally, we observed morphological change on hippocampus by HE staining and Nissl’s staining. As shown in Fig. 2c, d, pyramidal neurons in CA3 region were regularly arranged in control group and co-injection of miR-206 agomir with KA group, whereas that were irregularly arranged with a confused structure in KA group or KA + NC agomir group (Fig. 2c). And the results of Nissl’s staining showed that co-injection of miR-206 with KA protected KA-induced neuronal loss (Fig. 2d). These results indicated that expression of miR-206 suppressed the seizure and protected neurons in hippocampus from KA induced epilepsy.

Fig. 2.

Fig. 2

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Enforce expression of miR-206 in hippocampus protected neurons from KA-induced seizures. a Co-injection miR-206 agomir with KA reversed decrease of miR-206 in hippocampus. b Co-injection miR-206 agomir inhibited abnormal amplitude and spike frequency of brain waves induced by KA. c HE and Nissl’s staining of hippocampus

Expression of miR-206 decreased expression levels of CCL2 and IL-1β

We further tried to determine the mechanism through which miR-206 protected neurons in hippocampus from KA induced epilepsy. Previously studies showed that chemokines and cytokines mediated inflammatory response played an important role in KA-induced epilepsy (Cerri et al. 2017; Cerri et al. 2016; Xie et al. 2011). We guess miR-206 protected neurons by decreasing expression of chemokines and/or cytokines. Considering lipopolysaccharide (LPS) could enhance KA induced expression of chemokines/cytokines and seizure activity, we measured expression levels of CCL2 and IL-1β in hippocampus of rats which were co-injected with LPS (50 μg/kg i.p.) by qRT-PCR and western blot. The results showed that co-injection with LPS increased KA-induced expression of CCL2 and IL-1β, whereas applying miR-206 agomir eliminated KA and LPS induced expression of CCL2 and IL-1β (Fig. 3a, b). These results suggested that expression of miR-206 could decrease expression levels of CCL2 and IL-1β in hippocampus of KA-induced rats.

Fig. 3.

Fig. 3

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Expression levels of CCL2 and IL-1β in hippocampus in response to different combination of KA, LPS and miR-206 agomir. a Expression levels of CCL2 and IL-1β in hippocampus was determined by RT-qPCR. b Expression levels of CCL2 and IL-1β in hippocampus was determined by western blot, and densitometry of bolts analyzed by ImageJ for CCL2 and IL-1β were normalized to β-actin

CCL2 was a target of miR-206

It is well known that miRNA can decreasing expression of their target by directly binding to 3′UTR of the targets (Haenisch et al. 2015). Thus, we analyzed the potential miR-206 binding sites of CCL2 and IL-1β using RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) and found a binding site of miR-206 on 3′UTR of CCL2 (Fig. 4a). We further performed dual luciferase reporter assay and the result showed that mutations on miR-206 binding sites of CCL2 indeed abolished the inhibition of miR-206 mimics on the luciferase activity (Fig. 4b). Besides, transfection of miR-206 mimics decreased expression levels of CCL2 in cells which were determined by RT-qPCR and western blot (Fig. 4c). These results indicated that miR-206 down-regulated expression of CCL2 in vitro. Finally, we showed that miR-206 could abolish KA-induced expression of CCL2 in vivo using KA induced epilepsy model (Fig. 4d).

Fig. 4.

Fig. 4

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miR-206 down regulatedCCL2 by targeting to 3′UTR of CCL2. a Schematic representation of pmirGLO constructs containing the WT or mutations of 3′-UTR of CCL2 with miR-206 target sites. b, c Transfection with miR-206 mimics decreased expression of CCL2 determined by western blot and RT-qPCR Densitometry of bolts analyzed by ImageJ for CCL2 were normalized to β-actin. d, e Injection of miR-206 agomir restored the expression of CCL2 in KA-induced epileptic rats determined by western blot and RT-qPCR. Densitometry of bolts analyzed by ImageJ for CCL2 were normalized to β-actin

miR-206 suppressed the seizure and protected neurons through decrease of CCL2

To further confirm that miR-206 suppressed the seizure and protected neurons through decrease of CCL2, we constructed adeno-associated virus (AAV) encoding CCL2 and injected in combination with miR-206 agomir. The results showed that administration of miR-206 agomir in KA and LPS induced epilepsy rats decreased expression levels of CCL2 and IL-1β compared with rats administrated with NC agomir, while co-injection AAV-CCL2 with miR-206 agomir restored expression of CCL2 and IL-1β (Fig. 5a, b). In accordance with expression levels of CCL2 and IL-1β in the three groups, co-injection AAV-CCL2 recovered miR-206 agomir induced decrease of amplitude and spike frequency of brain waves (Fig. 5c). Moreover, HE and Nissl’s staining showed that co-injection AAV-CCL2 abolished miR-206 agomir mediated protection of neurons in hippocampus (Fig. 5d). These results confirmed that miR-206 mediated inflammatory response was critical to KA-induced epilepsy.

Fig. 5.

Fig. 5

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Enforced expression of CCL2 abolished protecting function of miR-206. a Amplitude and spike frequency of brain waves of rats under different treatment were analyzed. b, c Expression levels of CCL2 and IL-1β in hippocampus of rats under different treatment were measured by western blot and RT-qPCR. Densitometry of bolts analyzed by ImageJ for CCL2 were normalized to β-actin. d HE and Nissl’s stainging of hippocampus

Discussion

Epilepsy is one of the most common neurological diseases which affects about 50 million people worldwide. Although more than 24 antiepileptic drugs (AEDs) has been approved for use in the United States, one-third of the patients suffered from recurrence seizures due to no response to these drugs (Hanaya and Arita 2016; Sirven et al. 2012).Most of these drugs target against neuronal ion channels and both gamma-aminobutyric acid (GABA) and glutamate receptors to control neural excitation, however increasingly evidences show that inflammatory processes plays an important role in epilepsy (Srivastava et al. 2016). And miRNAs are very important to development and function of neurons. Dys-regulation of miRNAs has been found in various neurological diseases, such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), as well as epilepsy (Kye and Gonçalves 2014; Trivedi and Ramakrishna 2009). miRNAs could regulate neuronal inflammation, neuronal death and neuronal microstructure to regulate epileptogenesis related processes (Jimenezmateos and Henshall 2013). Previously studies have showed that miR-206 regulated tumorigenesis in various cancers, including breast cancer (Samaeekia et al. 2017), chondrosarcoma (Wang et al. 2017), head and neck squamous cell carcinoma (Koshizuka et al. 2017), lung adenocarcinoma (Chen et al. 2016) and gliomas (Yang et al. 2015). In this studies, we showed that miR-206 mediated neuronal inflammation was critical to KA-induced epilepsy.

In response to KA induced epilepsy, expression of miR-206 was specially decreased in hippocampus, but not in cortex, and in vitro studies showed decrease of miR-206 demonstrated a time and concentration dependent manner (Fig. 1). Besides, expression of miR-206 was also down-regulated in pilocarpine induced epilepsy models, and differential expression of miR-206 might be related with refractory to antiepileptic drugs (Moon et al. 2014). These results indicate miR-206 involves in epileptogenesis.

More and more studies suggest inflammatory processes in brain was common and crucial to the pathophysiology and development of seizures and epilepsy (Vezzani et al. 2011). Inflammatory mediators, such as IL-1β, CCL2, CCL3, CCL4, were upregulated in epilepsy (Rana and Musto 2018). Expression of CCL2 was upregulated in hippocampal neurons of mice with KA-induced seizures, which further activated STAT3 (signal transducer and activator of transcription 3, STAT3) to induce neuronal cell death. And lacking CCL2 or CCR2 in the mice attenuating seizure-induced degeneration of neurons in the hippocampal CA3 region confirmed the important role of CCL2 in epilepsy (Tian et al. 2017a). In this study, we showed that one possible mechanism through which miR-206 regulated epilepsy was decrease of neuronal inflammation by down regulation of CCL2. Up regulation of CCL2 resulted from decrease of miR-206 was a very important process in KA-induced epilepsy. Over expression of miR-206 by co-injection miR-206 agomir with KA alleviated rats from epilepsy, whereas enforced expression of CCL2 by AAV abolished mitigative effect of miR-206 agomir (Figs. 3, 5). Interestingly, we also found that changes of IL-1β which was not a target of miR-206 was in corresponding with changes of CCL2. This might cause by that expression of IL-1β was induced by CCL2 in status epilepticus (Cerri et al. 2016; Tian et al. 2017b).

Above all, we found that decreased expression of miR-206 played an important role in epilepsy, and enforced expression of miR-206 could alleviate seizures. Although the first-line antiepileptic drugs show very good anti-epileptic effect to prevent the occurrence of seizures, they have little effect on the underlying pathophysiology of epilepsy (Jimenezmateos and Henshall 2013). Our founding revealed the potential mechanism mediated by miR-206 in inflammatory induced epilepsy and these founding could provide potential therapy strategy. Since one miRNA could have multiple targets, more detailed research about function of miR-206 in epilepsy is needed.

Footnotes

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