GAO Yu-guang, MA Yu-juan, MO Qiong-ze, LIAO Yu-xiong, LIU Qi-qi, HE Qian-chao

1.The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023,China

Keywords:

ABSTRACT Objective:To explore the related mechanism of miR-34a affecting epilepsy.Methods:MicroRNA microarray chip and RT-PCR were used to analyze the expression of miR-34a in epileptic rats,and observe the relationship between miR-34a and seizure frequency and EEG.Predicted the target gene of miR-34a, used The dual-luciferase reporter gene assay was used to observe its binding to the target gene of miR-34a, the effect of overexpression and inhibition of miR-34a on the target gene, the effects of overexpression and inhibition of target genes on downstream factors.Results:Microarray chip and RT-PCR detection showed that miR-34a was highly expressed in epilepsy (P＜0.05), and miR-34a was positively correlated with seizure frequency (R=0.783, P=0.003), and inhibition of miR-34a inhibited the seizure frequency of epilepsy(P＜0.05).The online databases TargetScan and miRDB show that miR-34a and BCL-2 have a predicted binding region, and the dual-luciferase reporter gene experiment proves that the two have a binding relationship.When miR-34a was overexpressed, BCL-2 expression was down-regulated, and conversely, when miR-34a was inhibited, BCL-2 expression was up-regulated.NLRP1 and downstream related factors Caspase-3, Caspase-1, IL-1β and IL-18 were highly expressed in the brain tissue of epileptic rats(P＜0.05).When BCL-2 was inhibited,the expression levels of NLRP1 and downstream related factors were increased, and when BCL-2 was inhibited, the result was the opposite.Conclusion:The highly expressed miR-34a promotes epilepsy by inhibiting BCL-2 and activating the NLRP1 inflammasome.

1.Introduction

Epilepsy is one of the common brain diseases, with a high prevalence rate[1], The prevalence rate of epilepsy is about 0.5%to 1.0%; the case-fatality rate is (1.6 to 4.1)/100 000[2]After comprehensive treatment, some patients are mostly relieved, but 33% of the patients are still delayed, often develop, and develop into refractory epilepsy, the pathogenesis of epilepsy is not clear.Micro RNAs (miRNAs) is a group of small-fragment, singlestranded, and endogenous non-coding RNA, which participates in the pathogenesis of many diseases by identifying and binding to the 3′UTR region of the target mRNA[3].Combined with the target properties of miRNA, silencing or induction of miRNAexpression is a promising treatment for epilepsy[4].As reported in the literature,miR-34a can affect the development of epilepsy[5].To this end, this experiment is planned to explore the relevant mechanism of miR-34a affecting epilepsy, in orderto provide a basis for the treatment of epilepsy.

2.Materials and methods

2.1 Experimental animals

78 SD rats of SPF healthy 2-month-old males (63 in model group and 15 in control group), with an average weight of 201.34±5.71g,were purchased from Changsha Tianqin Biotechnology Co.,LTD(License No.: SCXK Xiang 2019-001).Rats were housed at 23-25℃ (temperature), 45%-55% (humidity), and had light times at 7:00 am-19:00 pm.

2.2 Model construction of epilepsy

Sixty-three rats were randomly selected to construct the epilepsy model.The epilepsy rat model was treated with lithium chloridepilocarpine hydrochloride[6], Rat were first given intraperitoneally with lithium chloride solution at 127 mg/kg, 20 h after atropine 1 mg/kg and 30 min before 40 mg/kg, Atropine toantagonize the peripheral cholinergic response caused by pilocarpine.Refer to the Racine′s[7] Observe behavioral changes in rats, level 0: no convulsions; level 1: only facial spasm; level 2: rhythmic nod and facial spasm; level 3: one forelimb clonus + rhythmic nod + facial spasm; level 4: hind limb standing + rhythmic nod + double forelimb clonus + facial spasm; level 5: double forelimb clonus + hind limb standing + facial spasm + rhythmic nod + fall.For the rats that did not reach grade 4 or above, 1/3 dose of pilocarpine was given every15 min, and the rats that still failed to meet the standard were considered as mold failure; If the seizures persisted for up to 1 h,diazepam could be given to terminate the epileptic seizure.

2.3 MicroRNA microarray chip analysis of rat brain tissue

Three epilepsy model rats and three control rats were randomly taken, severed after anesthesia, intact bilateral hippocampal tissue at the junction of the brain, grinding into slurry, treated with TRizol(Thermofisher, China, item number: 1218355), and RNA was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany).The miRNA amplification, subsequently hybridized with a Illumina expression profile microarray, labeled with cyanine 3 fluorescence labeled primers, again amplified, denatured, then hybridized with miRNA microarrays, incubated at 45-60 ℃, and finally imaging with laser excitation, and quantitative imaging of fluorescence intensity using the iScan system for expression analysis.Sequencing used P＜0.05, |log2FC| 1 as reference condition, and controls to differentially expressed miRNAs.

2.4 Target miRNA target gene prediction

Through the TargetScan database (http://www.targetscan.org/vert_72/) and the miRDB database (http://mirdb.org/) predicted the downstream target genes of miR-34a, followed by subsequent analysis.

2.5 Intervention and grouping

The remaining 60 epileptic rat models were randomly divided into 5 groups, 12 mice in each group: A, Model group (127 mg/kg of lithium chloride injection, 20 hours later, pilocarpine hydrochloride 40 mg/kg).B, The miR-34a overexpression group.C, and the miR-34a inhibition group.D, The BCL-2 overexpression group.E, The BCL-2 inhibition group.In the suppressor and overexpression groups, the target gene recombinant adenovirus vector (Shanghai Jikai Gene Medical Technology Co., LTD., miRNA U6 EGFP) was used, and 100 μL of the viral vector was injected twice a week for 3 weeks, so that the target gene was inhibited or overexpressed in rats.

2.6 Behavioral evaluation-seizure behavior record

At the end of the mold and after the end of the intervention began to recording rat seizure behavior, from 8 PM to 8 PM daily monitoring rat seizure behavior, monitoring 24 h, continuous monitoring for 3 d, monitoring time point for each group after the end of the mold,video recording number of seizures, finally calculate the average daily number of seizures in rats.

2.7 Monitoring of the EEG was performed

At the end of molding and 24 h after the intervention of each group, the stereoscopic brain positioning (Chengdu Instrument Factory, model ST-4ND) was used, with the midpoint of the anterior fontanelle of the experimental animal as the reference point, 1.2 cm beside the middle point of the anterior fontanelle, 0.6 cm backward,the depth was 4 cm, the trocar was placed, and the dental cement was fixed.The hippocampal electrodes were positioned as being shifted by 2.5 mm lateral to the median anterior fontanelle, 3.8 mm posterior, and 3.7 mm below the skull.The cerebellar reference electrode is positioned as the midline 1 mm after the skull.Rat EEG instrument (Shanghai Yuyan Scientific Instrument Co., LTD., No:EEG / EMG TETHERED SYSTEMS) was embedded in the above positioning.The tracing condition was 0.3 and gain of 50 μV.Rat EEG activity was monitored by continuous power spectrum and recorded by computer.

2.8 RNA extraction and reverse transcription polymerase chain reaction

Hippocampal tissue was ground for total RNA using TRIzol reagent (Invitrogen) and obtained using the miRNA Reverse Transcription kit (Cat.no.4,366,596 l; Applied Biosystems, Foster City, CA, USA) for reverse transcription.Expression of the target gene was quantified using TaqMan Universal Master Mix II (cargo number 4440038; Applied Biosystems).The experiment was set to be pre-denatured into a single chain at 94 ℃, and repeated as a template for 35 cycles (30 s at 94 ℃ and 45 s at 55 ℃).Finally, the ratio of the target gene and the reference gene (GAPDH) is indicated by Ct; 2-ΔΔCTRepresents the measured relative RNA expression

2.9 Cell culture

HEK293T cells were purchased from the American Type Culture Collection (ATCC) with Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (Gibco) at 5% CO2And 37 ℃ in the incubator.

2.10 Cells were transfected

The BCL-2 overexpression vector and the negative control were both provided by Shanghai Jikai Gene Medical Technology Co., Ltd.Vectors were imported into HEK293T using Lipofectamine 3 000 (Invitrogen,Carlsbad, CA, USA) following the manufacturer′s protocol.

2.11 Luciferase reporter assay

A Luciferase reporter containing a wild-type or mutant plasmid was constructed using the psi-CHECK2 vector (Promega, Madison, WI,USA).Luciferase vector containing the plasmid of the pcDNA3.1-BCL-2-untranslated region was cotransfected into HEK293T cells with a miR-34a mimic or a negative control using Lipofectamine 2 000 (Invitrogen), and 10 ng of the sea renilla luciferase reporter was used as an internal control.After 48 h, the cells were harvested and lysed.Luciferase activity was measured using a Dual-luciferase reporter assay system(Promega).

2.12 The Western blot experiment

Rat hippocampal tissue was lysed at 4 ℃ for 5 min after using the protein lysate, centrifuged at 1 000 rpm for 10 min, the tissue was deprecipitated, and the supernatant was retained.Protein concentration was measured for each sample.Then electrophoresed,the glue concentration was 8% and 50 μg protein sample was added to each lane.After bromoophl blue reaches the bottom of the separation glue, the membrane was transferred.After membrane transfer, the PVDF membrane was placed in the blocking solution containing 5% skim milk for 1 hour at room temperature, adding primary antibody (BCL-2, NLRP1, Caspase-3, Caspase-1, IL-1β,IL-18), and incubated overnight for 4 ℃ overnight.After washing PVDF membranes three times with WB wash solution the next day, secondary antibodies were added and incubated for 1h at room temperature.The WB wash solution washed the PVDF film three times, added 3 mL of chemiluminescence solution (ECL), and then performed the photosensitive film imaging.The gray values of the signal were determined using the ImageJ image analysis system.

2.13 Statistical Methods

Using SPSS 23.0, the measurement data are described by (±s).When the two groups meet the normal distribution, t-test is used,single-variance analysis is used for comparing the three groups, and non-parametric method.P＜0.05 is statistically significant.

3.Results

3.1 The miR-34a is upregulated in the brain tissue of epileptic rats

A total of 40 differentially expressed miRNAs s were selected in epilepsy models, where miR-34a was highly expressed in epilepsy models (Figure 1A).R T-PCR detection also confirmed that miR-34a was highly expressed in the hippocampus of epilepsy model rats (Figure 1B, Table 2), and miR-34a expression was positively correlated with seizure frequency (R:0.783, P=0.003, Figure 1C).The EEG of epileptic rats had epileptic abnormal sharp spike or spike waves, which were reduced when miR-34a was inhibited(Figure 1D).In addition,overexpressing, overexpressing and inhibiting miR-34a increased and reduced the frequency of seizures,respectively (Figure 1E, Table 3).

Tab 1 Primer Sequences

3.2 BCL-2 is a potential target of miR-34a

While miR-34a has a binding region with BCL-2 (Figure 2A),the wild-type group of luciferase reporter plasmid activity was reduced compared to the mutant miR-34a group (Figure 2B, Table 3).BCL-2 expression was downregulated in epileptic rats compared to controls (Figure 2C, Table 4), contrary to miR-34a expression.The mRNA expression of BCL-2 was upregulated afterinhibiting miR-34a expression, with the opposite result observed when miR-34awas overexpressed (Figure 2D, Table 5), and this result was also obtained by immunoblot experiments (Figure 2E).In addition,the overexpression of BCL-2effectively reduced the frequency of seizures, while the inhibition of BCL-2 was reversed (Figure 2F,Table 6).

Tab 2 Relative expression levels of miR-34a in the control and model groups(n=12,±s)

Tab 2 Relative expression levels of miR-34a in the control and model groups(n=12,±s)

group expression level of miR-34a control group 0.97±0.19 model group 1.93±0.23 t 11.18 P＜0.001

Tab 3 Frequency of seizures in each group(n=12, ±s)

Tab 3 Frequency of seizures in each group(n=12, ±s)

group Seizure seizure frequency The miR-34a overexpression group 21.33±3.44 model group 16.92±3.45 The miR-34a inhibition group 13.50±2.64 F 18.039 P＜0.001

Tab 4 The miR-34a wild-type and mutant luciferase activities( ±s )

Tab 4 The miR-34a wild-type and mutant luciferase activities( ±s )

group Luciferase activity Wild-type miR-34a 0.55±0.17 The miR-34a mutant type 2.33±0.34 t 14.81 P＜0.001

Tab 5 Relative expression levels of BCL-2 in each group(n=12,±s )

Tab 5 Relative expression levels of BCL-2 in each group(n=12,±s )

group expression level of mi R-34a control group 1.97±0.21 model group 0.98±0.19 The miR-34a overexpression group 0.66±0.23 The miR-34a inhibition group 1.41±0.35 F 11.023 P＜0.001

Tab 6 Frequency of epileptic seizures in each group(n=12,±s )

Tab 6 Frequency of epileptic seizures in each group(n=12,±s )

group Seizure seizure frequency The BCL-2 overexpression group 11.75±2.73 model group 16.92±3.45 The BCL-2 inhibition group 22.42±3.45 F 25.84 P＜0.001

Fig 1 mi R-34a is involved in the pathogenesis of epilepsy

3.3 BCL-2 regulates the NLRP1 inflammasome and its downstream associated inflammatory factors

The mRNA expression of NLRP1, Caspase-3, Caspase-1, IL-1β,and IL-18was all higher than that of the control group (Figure 3A).BCL-2 overexpression inhibited the expression of these factors in the hippocampus of the epilepticrat model (Figure 3B), and BCL-2 showed opposite results (Figure 3C, Table7).

Fig 3 Effects of BCL-2 on the NLRP1 and its downstream inflammation-related factors

Tab 7 Relative expression levels of factors in each group

Fig 2 miR-34a regulates BCL-2 expression

4.Discussion

miRNAs plays an important role in the occurrence and development of epilepsy, but the specific molecular mechanism has not been defined[8].The miR-34a, the first miRNA shown to regulate apoptosis, regulates neurodevelopmental processes[9].Yan Yan et al[10] Studies have confirmed that miR-34a is highly expressed in epileptic rats.HuK class[11] It was confirmed that miR-34a increased with increasing seizure frequency in the BM-induced epilepsy model.Li LAN et al[12] It was also confirmed that miR-34a in CSF and serum in epileptic patients.In this study, we found that miR-34a was highly expressed in the hippocampus of the epilepsy model rat, which was positively correlated with the seizure frequency,consistent with the above study report.

How miR-34a regulates epilepsy still needs to be deeply studied.Here, the potential target gene of miR-34a is BCL-2 through the online database TargetScan and miRDB, and the literature study suggests that the miR-34a/BCL-2 axis is associated with multiple diseases[13-15], And miR-34a can inhibit BCL-2 expression[16]However, it has not been reported in epilepsy.The dual luciferase reporter experiments proved that BCL-2 is probably a binding factor of mi R-34a.Further studies found that BCL-2 mRNA expression of BCL-2 has suppressed miR-34a expression, and promoting BCL-2 expression could effectively showed that miR-34a can target and negatively regulate BCL-2 to regulate seizure frequency.BCL-2 is involved in neural cell apoptosis[7], Mitochondrial damage[18]Other biological processes, BCL-2 is closely related to epilepsy,and other studies have shown that BCL-2 inhiinhibiting apoptosis and inflammatory response[19-20]To improve mitochondrial dysfunction[21]And thus reducing the frequency of seizures.

In recent years, the research of epilepsy and inflammatory factors has attracted much attention, and a large number of studies have confirmed it[22-23] Various inflammatory mediators including apoptotic factors, interleukin and other seizures can induce seizures.BCL-2 is an anti-apoptotic protein, which mainly suppresses cell apoptosis from various causes, and BCL-2 was found to inhibit the activation of the NLRP1 inflammasome[24] To reduce neural cell apoptosis and inflammation and reduce seizure onset, in order to verify whether BCL-2 regulates NLRP1 inflammasome to affect the development of disease in epilepsy rats, our further study found that NLRP1 and its downstream mRNA of Caspase-3, Caspase-1, IL-1β and IL-18 were all expressed in high state in the brain tissue of epilepsy model rats.The expression of NLRP1 and downstreamrelated factors, which were downregulated and inhibited by BCL-2 over-expression, was reversed in the brain tissues of rats.Mohseni-Moghaddam Pet al[25-26] Studies have also confirmed that the expression of NLRP1 and Caspase-1 was increased in the hippocampus of the epileptic rat model, and Zhou Ruhan et al[27]Studies have shown that after Caspase-1 activation, the inflammatory response will be initiated, and the activated Caspase-1 enzyme will also induce increased IL-1β and IL-18 expression, aggravating the nerve cell inflammatory response, and thus inducing recurrent seizures.Alternatively, as confirmed by Wang YC et al[28-30]The NLRP1 inflammasome is also involved in the nerve cell inflammatory response, which induces nerve cell and mitochondrial damage after epilepsy by promoting Caspase-3 expression.When rat NLRP1 was knocked down, Caspase-1 and IL-1β expression were also decreased in the brain tissue of ratswith temporal lobe epilepsy,when the seizure frequency was reduced[31-32].All of the above studies showed that BCL-2 affects epileptic seizures by mediating NLRP1 and its downstream inflammatory cytokines.

In conclusion, our study found that the expression situation of miR-34a was positively correlated with the frequency of seizure onset, and that miR-34a induced epilepsy by inhibiting BCL-2 and activating the NLRP1 inflammasome, thus producing downstream inflammatory sublinks.Therefore, inhibition of miR-34a expression may be an important target for future treatment of epilepsy.

The Conflict of Interest Statement:

The authors declare that there are no conflicts of interest related to the manuscript.

Author contribution degree description:

Gao Yuguang: research data statistical analysis, article writing,overall experiment planning and arrangement; Ma Yujuan: animal experiment, the epilepsy model production, animal breeding; Mo Paze: collect experimental data, immunoblot detection, animal breeding; Liao Yuhao: PCR testing, collect experimentaldata;Liu Qiqi: EEG monitoring, animal behavior data collection; He Qianchao:project research idea designer, project leader, develop the overall research objectives.