Detection of Deregulated miRNAs in Childhood Epileptic Encephalopathies

The term “epileptic encephalopathy” is used to describe a possible relationship between epilepsy and developmental delay. The pathogenesis of developmental encephalopathies, independent of epilepsy, can be defined by genetic control mechanisms. The aim of this study was to investigate the use of miRNAs as serum biomarkers for the determination and discrimination of epileptic encephalopathies. Whole blood samples obtained from 54 individuals in 2 groups designated as epileptic encephalopathy patients’ group (n = 24) and healthy controls (n = 30) were included in this study. The expression levels of 10 miRNAs were determined using qRT-PCR. After the determination of expression levels, the correlation of upregulated miRNA levels and Ki67 index was calculated using Pearson correlation test. The comparison of epileptic encephalopathy patients’ group with healthy controls revealed the upregulation of one miRNAs (hsa-miR-324-5p) and downregulation of three miRNAs (hsa-miR-146a-5p, hsa-miR-138-5p, hsa-miR-187-3p). It has been determined that miRNAs with altered expression are an important factor in the formation of epileptic seizures and seizure-induced neuronal death. The fact that processes that play a key role in epiloptogenesis are under the control of miRNAs causes miRNAs to become meta-controllers of gene expression in the brain. We thought that further studies are needed to prove that especially hsa-miR-146a-5p, hsa-miR-138-5p, and hsa-miR-187-3p can be used as epileptic encephalopathy biomarkers. The detection of disease-specific miRNAs could contribute to the development of precision treatments.


Introduction
Epilepsy is a common neurological disorder in childhood defined as seizures that occur because of neuronal discharge in a specific region of the brain (Li et al. 2020). The incidence of the disease in children varies between 33 and 82 per 100,000 per year (Wu et al. 2019). Drug-resistant epilepsy (DRE) is defined as the persistence of seizures despite adequate anti-epileptic treatment with two or more anti-epileptic drugs at efficacious daily doses, alone or in combination, and affects approximately 30% of children with epilepsy. Nowadays, it has been defined as not pharmacoresponsive epilepsies. The most severe forms of drug-resistant epilepsy usually begin in childhood (Patel and Mercimek-Mahmutoglu 2016). In patients with epilepsy related to a genetic etiology, drug resistance is particularly frequent, especially in those with severe developmental and epileptic encephalopathies (Guery and Rheims 2021).
Epileptic encephalopathy is defined by the presence of frequent epileptiform activity that causes slowing or regression of developmental skills. It is known that many of the epileptic encephalopathies have a molecular genetic basis. The fact that genetic etiology alone can cause developmental impairment means that individualized treatments should be developed to prevent the devastating consequences of this disease group (Scheffer and Liao 2020). According to the 2010 International League Against Epilepsy (ILAE) classification and terminology commission, epileptic encephalopathy is defined as conditions in which epileptic activity causes serious cognitive and behavioral disorders and may worsen over time, beyond what is expected from the existing pathology (Berg et al. 2010). Childhood EE are classified as infantile spasms, Dravet syndrome, Lennox-Gastaut syndrome, epileptic encephalopathy with electrical status during slow sleep (ESES), and Landau-Kleffner syndrome, etc. The common feature of these syndromes is that they are generally resistant to antiseizure drugs (ASD) (Engel 2001). The response to (ASD) is generally poor despite highdose polytherapy. The most common application used in the treatment of EE patients is adrenocorticotropic hormone (ACTH). However, ACTH causes side effects with high morbidity and mortality, such as hyperglycemia, hypertension, immunodeficiency, and iatrogenic Cushing's syndrome (Khan and Al Baradie 2012).
Understanding what controls gene expression may open new avenues for the treatment or prevention of epileptic encephalopathy (Fisher et al. 2014). Therefore, the development of new treatment targets and strategies in epileptic encephalopathies will reduce the risk of mortality and morbidity due to seizures and drugs used by individuals with this disease. For this purpose, identifying the relevant pathways and molecular mechanisms that coordinate gene expression is crucial for a better understanding of the pathogenic process and the development of new therapeutic approaches. microRNA (miRNA) is a class of small (19-25 nucleotide), single-stranded, endogenous, non-protein-coding short RNA molecules that regulate gene expression either by promoting mRNA degradation or attenuating protein translation at the post-transcriptional level (Reschke and Henshall 2015). miRNAs are initially transcribed by polymerase II (Pol II) as primary transcripts (pri-miRNA), processed into pre-miRNA, and finally converted to mature miRNA by RNases called Drosha and Dicer, respectively.
The functional strand of mature miRNA is loaded into the RNA-induced silencing complex (RISC) containing the Argonaute 2 (Ago-2) protein. The RISC-loaded miRNA is then directed to silencing the target mRNA via mRNA degradation or translation inhibition. A given miRNA can have several binding sites to the same mRNA, and a single mRNA can be targeted by more than one miRNA, thus producing stronger effects. In line with this view, a single miRNA can regulate the expression of hundreds of genes, thus presenting important effects on cellular functions (Esquela-Kerscher and Slack 2006). They mainly function to lower protein levels in cells through sequence-specific binding to target mRNAs, leading to transcript degradation or translational repression.
More than 50% of the miRNAs identified are expressed in the brain. The brain has a variety of miRNAs that are crucial for the establishment and maintenance of normal development and cell phenotype. Acute and chronic nervous system diseases, including epilepsy, are associated with dysregulation of key components of the miRNA biogenesis pathway and altered expression of miRNA. miRNAs are implicated in many brain functions important for epileptogenesis, including cell death, neurogenesis, and synaptic plasticity (Karnati et al. 2015). Studies on associating the development of epilepsy with miRNA have gained momentum in the last 10 years. Large-scale miRNA profiling studies that characterize changes in the expression of different miRNAs are carried out, especially in animal models and subsequent experimental studies with human blood and tissue samples (Mooney et al. 2016). Recently, functional studies in rodents have shown that miRNAs can have potent effects on brain excitability, seizures, and epilepsy. It has been reported that targeting miRNAs, which play a key role in epilepsy, suppresses or exacerbates seizures and alters brain excitability, which has a potential for miRNA-based therapeutics in epilepsy (Reschke and Henshall 2015).

Study Design
A total of 54 samples from 30 healthy controls and 24 epileptic encephalopathy patients were included in the study. Blood samples were taken from volunteers who were prediagnosed in Health Sciences University, Dr. Behçet Uz Children's Training and Research Hospital, Department of Pediatric, Division of Pediatric Neurology outpatient clinics, aged between 1 month and 18 years, who underwent electroencephalogram (EEG), brain magnetic resonance imaging (MRI), and metabolic tests and were diagnosed with EE. Control group samples were taken from healthy individuals matched for age and sex. The study was approved by the Clinical Research Ethics Committee of Izmir Dr. Behçet Uz Children's Training and Research Hospital (Project No: 2019/084, Ethical Approval No: 2019/07-07). After all, individuals included in the study or their parents read and signed the informed consent form, and 2 ml peripheral blood samples were taken into EDTA tubes for miRNA isolation and centrifuged at 3000 rpm for 10 min for serum separation from blood. Serum samples were stored at − 80 °C until the study was carried out and transferred to the Health Sciences University Medical Biology Department by the cold chain for miRNA isolation.
miRNA Extraction and cDNA Synthesis miRNA isolation was performed from serum samples using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany, Cat. No: 217204) according to the manufacturer's instructions. Next, cDNA synthesis from miRNAs was performed using the miRCURY LNA RT Kit (Qiagen, Hilden, Germany, Cat. No: 339340) according to the manufacturer's instructions. To determine the purity and concentration of cDNA samples, cDNA control PCR and melting curve analysis were performed.

qRT-PCR
Pre-designed primers were used for real-time PCR to determine miRNA expression levels of all samples (Table 1). In total, a gene panel was constructed with specific primers and one housekeeping primer for 10 different miRNAs (miR-CURY LNA miRNA Custom PCR Panels, Qiagen, Hilden, Germany). Real-time PCR reactions were performed in triplicate on a Light-Cycler 480 II (Roche) instrument following the manufacturer's protocol using the miRCURY LNA SYBR Green PCR Kit (Qiagen,Hilden,Germany,Cat. No: 339346). Thermal cycling conditions were performed at 95 ºC for 10 min, then at 95 ºC for 15 s, at 60 ºC for 1 min, and at 72 ºC for 10 s for 45 cycles. PCR specificity was confirmed by melting curve analysis.

Statistical Analysis
SPSS 25.0 was used for statistical analysis. The significance of miRNA expression levels was calculated by performing a t test on 2-Δct values for each different miRNA. For the analysis of continuous variables, the Shapiro-Wilk test and Levene's test was facilitated to investigate the distribution and variance homogeneity. Parametric continuous values were compared with t test and ANOVA, while non-parametric variables were compared with Mann-Whitney U and Kruskal-Wallis tests. Categorical variables were analyzed by chi-square tests. Significant correlations between the expression data and clinical parameters were assessed by calculating the Pearson correlation coefficient. The obtained data were analyzed with an online software program (miRCURY LNA miRNA PCR Panels and Assay) developed by Qiagen GeneGlobe Data Analysis Center. The expression levels of each miRNA were evaluated using the Ct (Threshold Cycle) value. These results were normalized for each sample using the SNORA66 housekeeping gene (∆Ct). The relative amount of each miRNA to SNORA66 was described by the equation ∆Ct = (Ct miRNA − Ct SNORA66). Fold changes between control and patient were calculated as ∆∆Ct. The N-fold differential expression of the miRNA gene of the patient sample compared with the control was expressed as 2 − ∆∆Ct. The mean values of fold difference for epileptic encephalopathy patients were compared with the mean values obtained from control samples. In this study, miRNAs with fold change ≥ 2 were considered deregulated.

Demographic and Clinical Characteristics of the Study Population
The demographic and clinical characteristics of both case and control groups were summarized in Table 2. The mean ages for EE patients and healthy control groups were 0.1 ± 8.77 and 0.1 ± 11.02, respectively. When the EEG findings of the patients were examined, 7 patients showed Lennox-Gastaut syndrome, 2 patients showed generalized epileptic activity, 2 patients showed Focal ESES, 6 patients showed focal epileptic activity, and 5 patients showed modified hypsarrhythmia. When the patients were evaluated in  terms of developmental and behavioral disorders, mental retardation was found in 18 patients, growth retardation in 4 patients (cognitive retardation in 1 patient, global growth retardation in 1 patient, growth, and development retardation in 2 patients), autism in 1 patient, and normal psychomotor development in 1 patient.

miRNA Deregulation in Epileptic Encephalopathy Patients
To determine the expression levels of 10 different miR-NAs (Table 3) that are thought to be deregulated in epileptic encephalopathy patients compared to healthy controls, serum miRNA expression levels were determined by the qRT-PCR method in 30 healthy controls and 24 epileptic encephalopathy patients.

Discussion
Children with EEs are in the risk group in terms of developmental problems, mental status disorders, and comorbidities such as physical problems. The cognitive outcome is not only related to the cause but also appropriate treatments, such as hormonal therapy or early surgical treatment in cases with focal cortical dysplasia (Sisodiya 2020). In our study, only one patient had normal neuromotor development, and most of the remaining patients had mental motor retardation, as well as any autism, growth and developmental retardation, global developmental retardation, and cognitive retardation.
In recent epilepsy genetics studies, miRNAs are important regulators of gene expression (Mooney et al. 2016). The failure of preclinical trials targeting genes encoding proteins that are effective in the development of epilepsy to prevent epileptogenesis has contributed to the prominence of miRNAs (Pitkanen and Lukasiuk 2011). The fact that processes that play a key role in epileptogenesis, such as neuronal death, gliosis, inflammation, and reorganization of neuronal networks, is under the control of miRNAs, causing miRNAs to become meta-controllers of gene expression in the brain (Dogini et al. 2013). Therefore, researchers are trying to determine their potential as biomarkers by manipulating miRNAs in vivo to understand the function of miRNAs that play a role in epilepsy. It has been determined that miRNAs with altered expression are an important factor in the formation of epileptic seizures and seizure-induced neuronal death . Approximately 100 miRNAs with varying expression levels were detected in miRNA profile screening studies performed with tissues isolated from the hippocampus resected from patients with temporal lobe epilepsy (Kan et al. 2012;McKiernan et al. 2012). According to the results of these studies, miR-146a and miR-132 are upregulated, while miR-30a/b, miR-138, miR-324, miR-330 (Bot et al. 2013a, b), andmiR-187 (Gorter et al. 2014) are downregulated. The first microRNA shown to regulate susceptibility to seizures was miR-134, which has an anticonvulsant effect when inhibited with antisense oligonucleotides prior to a seizure-inducing treatment . They detected that the expression level of miR-134 was increased in epilepsy and silencing of miR-134 caused a significant reduction in induced and spontaneous seizures. Limk1, the target gene of miR-134, is an important protein that plays a role in the control of the dendritic structure, which is a critical contact point for excitatory transmission in the central nervous system. In recent years, microRNAs such as miR-132, miR-146a, miR-34a, miR-128, miR-324-5p, and miR-124 have also been shown as positive or negative regulators of neuronal hyperexcitability (Tiwari et al. 2018). miR-146a has been shown to be strongly expressed in the central nervous system (CNS) and plays an important role in the regulation of the innate immune response induced in these tissues in association with neurodegenerative disorders. Aronica et al. (2010) is the first to report altered expression pattern of miR-146a in epileptic rats and temporal lobe epilepsy patients. Recently, miRNA expression profile analyses of samples under epilepsy conditions showed that miR-146a is significantly upregulated in the hippocampus of both the TLE rat model and epilepsy patients, and miR-146a also interacts with IL-1β at different stages of epilepsy. It has been proven that miR-146a has a key role in the cell cycle control, acquisition of neuronal identities, differentiation of neural cells, and regulation of the immune responses in the CNS. Its abnormal expression is among the most common events in the pathogenesis of neurological disorders (Nguyen et al. 2018;Mei et al. 2011). As can be seen, conflicting results regarding miR-146a expression have been reported in various publications. In our study, miR-146a showed downregulation. We explain this situation with the effect of miR-146a on the regulation mechanism of immune response. NF-κB signaling pathway activation induces miRNA gene expression. Negative regulation of this pathway activity is performed by miR-146a (Taganov et al. 2006). This miRNA acts as a post-transcriptional negative regulator in the immune cells by affecting TLRs, especially TLR4, and targeting IL-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6 adapter molecules (Doxaki et al. 2015). The lack of miR-146a expression is associated with impaired Treg function, loss of peripheral T lymphocyte tolerance, and autoimmunity occurrence (Boldin et al. 2011;Lu et al. 2010). We are investigating the miRNA expression levels of these target genes (IL-1, NF-kB, IRAK1, and TRAF6) related to the immune response, which we think may be relevant, as part of our ongoing project. An article made with cell lines published by Qin et al. in July 2021 reported that the miR-138-5p gene is a miRNA targeting caspase-3 to control apoptosis (Qin et al. 2021). In this study, it was reported that miR-138-5p is directly associated with the proliferation and survival of human multiple myeloma cells. We think that miR-138-5p, which was found to be downregulated in our study, triggers apoptosis, negatively affects cell survival, and causes damage to many brain functions that are important for epileptogenesis. Several studies have shown that miR-187 is downregulated in patients with TLE. Kretschmann et al. (2015) found a significant downregulation of miR-187 expression in the 28-day pilocarpine-induced SE in mice. Bot et al. (2013a, b) reported a downregulation of this miRNA 30 days after SE induced by amygdala electrical stimulation. Gorter et al. (2014) detected a significant downregulation of miR-187 at 3-4 months following status epilepticus induced by electrical stimulation of the angular beam in rats. Several studies have suggested a link between miR-187 and chronic human inflammatory diseases (Holliday et al. 2011;Suojalehto et al. 2014). In animal studies and clinical observations, inflammation-associated miRNAs have been found to be deregulated during the chronic phase of TLE (Omran et al. 2012;Ashhab et al. 2013). Evidence obtained from both in vitro and in vivo experiments indicated that miR-187-3p is involved in CFC memory (Zhao et al. 2020). In an article published in 2017 by Suya Zhang et al. the potential binding sites of miRNAs and their target genes were analyzed. rno-miR-187-3p was downregulated in stimulated groups compared to controls. Seven distinct genes were identified in the genome as predicted target genes of rno-miR-187-3p. The results obtained in our study overlap with these results (Zhang et al. 2017). In an article by Dakterzada et al. in Alzheimer's patients and published in 2020, miR-324-5p expression was found to be upregulated (Dakterzada et al. 2020). Also, according to Gross et al. (2016) study, inhibiting miR-324-5p is neuroprotective and increases the delay to seizure onset in mouse models of status epilepticus. The upregulation of the miR-324-5p gene, which is known to cause cortical dysplasia, which is reported to be upregulated in many cancer types such as chronic pancreatitis, colon cancer, and breast cancer, was also found to be upregulated in our study, in line with the literature (Kalser and Cross 2018;Wang et al. 2017).
Developmental delay in children with epilepsy may be the result of intense epileptiform activity (seizures and EEG abnormalities) or a combination of related factors. However, these seizures do have an impact on the quality of life, as well as the patient's safety, and as such need intervention. Therefore, the current "International League Against Epilepsy" classification has defined three electroclinical conditions: "developmental encephalopathy," "epileptic encephalopathy," and "developmental and epileptic encephalopathy" (DEE). Many biological pathways may play a role in the pathogenesis of DEEs. DNA repair, transcriptional regulation, axon myelination, metabolite and ion transport, and paroxysmal function may all be involved in DEE (Hamdan et al. 2017;Raga et al. 2021).
Although miRNA studies have been performed in adulthood, especially in mesial temporal lobe epilepsy, this rate is very limited in childhood epilepsy. In recent years, the fields of RNA-based therapy have advanced considerably, and miRNAs offer powerful new features in this field. A study on miRNA expression only in cases with EE was not found in the literature review. Many biological pathways may play a role in the pathogenesis of DEEs. Among these, channelopathies and synaptic dysfunctions are the most common (He et al. 2019). Causes of DEEs include disruption of other cellular functions, DNA repair, transcriptional regulation, axon myelination, metabolite, and ion transport, and peroxisomal function (McTague et al. 2016). Understanding the genetic causes of EE is essential for targeted therapies. By uncovering the pathogenetic mechanism and neurobiological processes, it may be possible to improve not only seizure control but also developmental outcome and disease-related comorbidities. However, there is a need to develop new and safe therapeutic drugs, particularly through preclinical research, to evaluate how these therapeutic interventions can be administered safely without causing unwanted side effects such as the currently widely prescribed ASDs (Tecuapetla et al. 2020).

Conclusion
Determining the molecular mechanisms of these miRNAs, which play a role in the development of EE, is very important for the development of precision treatments. In this study, we detected deregulated expression levels of four of the miRNAs. We hope that our study will shed light on further studies and that these disorders can be treated better with supportive research.