Identification of miRNA expression profiles in human mesial temporal lobe epilepsy with hippocampal sclerosis
Petra BencúrováJiří BalounKateřina MusilováKarol PálNikola TomJakub HynštLenka RadováMartin PailEva BrichtováMarkéta HermanováŠárka Pospı́šilováMarek MrázMilan Brázdil
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Purpose: The primary aim of this study was to identify specific miRNAs with aberrant expression in hippocampal tissue of mesial temporal lobe epilepsy (mTLE) patients with hippocampal sclerosis (HS). Method: We analyzed miRNA isolated from paraffin embedded tissue slices of hippocampal complex from 22 mTLE +HS patients (14 left-sided, 8 right-sided) and 9 post mortem controls. To characterize the miRNAs highly prone to be involved in the pathological changes in the mTLE+HS brain tissue we performed the expression analysis combining the next generation sequencing (NGS) and novel specific technique for miRNA quantification miQPCR. Results: By both methodical approaches we confirmed significant upregulation of miR-129-1-3p, 129-2-3p, 142-3p, 191-5p, 193b-3p, 195-5p, 374b-5p, 451a, and 490-3p in mTLE+HS patients. The levels of miRNA-184 were significantly higher in control subjects. Differential expression levels in mTLE+ HS patients were identified also in the case of 10 miRNA species never considered to play a role in epilepsy before. Conclusion: Analytical approach combining NGS with miQPCR used for the first time for analysis of human epileptic foci led to reliable identification of miRNA with altered expression in mTLE +HS patients.Keywords:
Hippocampal sclerosis
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Abstract Background To investigate the effect of long-term febrile convulsions on gene expression in mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) and explore the molecular mechanism of MTLE-HS. Methods Microarray data of MTLE-HS were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between MTLE-HS with and without febrile seizure history were screened by the GEO2R software. Pathway enrichment and gene ontology of the DEGs were analyzed using the DAVID online database and FunRich software. Protein–protein interaction (PPI) networks among DEGs were constructed using the STRING database and analyzed by Cytoscape. Results A total of 515 DEGs were identified in MTLE-HS samples with a febrile seizure history compared to MTLE-HS samples without febrile seizure, including 25 down-regulated and 490 up-regulated genes. These DEGs were expressed mostly in plasma membrane and synaptic vesicles. The major molecular functions of those genes were voltage-gated ion channel activity, extracellular ligand-gated ion channel activity and calcium ion binding. The DEGs were mainly involved in biological pathways of cell communication signal transduction and transport. Five genes ( SNAP25, SLC32A1, SYN1, GRIN1, and GRIA1 ) were significantly expressed in the MTLE-HS with prolonged febrile seizures. Conclusion The pathogenesis of MTLE-HS involves multiple genes, and prolonged febrile seizures could cause differential expression of genes. Thus, investigations of those genes may provide a new perspective into the mechanism of MTLE-HS.
Hippocampal sclerosis
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Deep brain stimulation (DBS) has seizure-suppressing effects but the molecular mechanisms underlying its therapeutic action remain unclear. This study aimed to systematically elucidate the mechanisms underlying DBS-induced seizure suppression at a molecular level.We established a macaque model of mesial temporal lobe epilepsy (mTLE), and continuous high-frequency hippocampus DBS (hip-DBS) was applied for 3 months. The effects of hip-DBS on hippocampus gene expression were examined using high-throughput microarray analysis followed by bioinformatics analysis. Moreover, the microarray results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses.The results showed that chronic hip-DBS modulated the hippocampal gene expression. We identified 4119 differentially expressed genes and assigned these genes to 16 model profiles. Series test of cluster analysis showed that profiles 5, 3, and 2 were the predominant expression profiles. Moreover, profile 5 was mainly involved in focal adhesion and extracellular matrix-receptor interaction pathway. Nine dysregulated genes (Arhgap5, Col1a2, Itgb1, Pik3r1, Lama4, Fn1, Col3a1, Itga9, and Shc4) and three genes (Col1a2, Itgb1, and Flna) in these two pathways were further validated by qRT-PCR and Western blot analyses, respectively, which showed a concordance.Our findings suggest that hip-DBS could markedly reverse mTLE-induced abnormal gene expression. Findings from this study establish the basis for further investigation of the underlying regulatory mechanisms of DBS for mTLE.
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Abstract MicroRNAs (miRNAs) have been proposed as biomarkers for cancer and other diseases due to their stability in serum. In epilepsy, miRNAs have almost been studied in brain tissues and in animals' circulation, but not in circulation of human. To date, a major challenge is to develop biomarkers to improve the current diagnosis of epilepsy. The aim of this study was to evaluate whether circulating miRNAs can be used as biomarkers for epilepsy. We measured the differences in serum miRNA levels between 30 epilepsy patients and 30 healthy controls in discovery and training phases using Illumina HiSeq2000 sequencing followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays. The selected miRNAs were then validated in 117 epilepsy patients and 112 healthy controls by qRT-PCR. Let-7d-5p, miR-106b-5p, -130a-3p and -146a-5p were found up-regulated, whereas miR-15a-5p and -194-5p were down-regulated in epilepsy patients compared to controls (P < 0.0001). Among these miRNAs, miR-106b-5p had the best diagnostic value for epilepsy with 80.3% sensitivity and 81.2% specificity. Circulating miRNAs were differentially regulated in epilepsy patients as compared with controls. MiR-106b-5p may serve as a novel, noninvasive biomarker to improve the current diagnosis of epilepsy.
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Mesio temporal lobe epilepsy (MTLE) syndrome is the most common form of intractable epilepsies. Meanwhile, seizures are common in patients with cancer as a consequence of brain tumors, including brain low-grade gliomas (LGG). However, the underlying molecular mechanisms of MTLE remain poorly understood. Also, the relationship between MTLE and LGG needs our attention. In this study, we aimed to investigate the hub genes and potential mechanism in MTLE, and the relationship between MTLE and LGG, the gene expression profiles (GSE88992) were downloaded from the Gene Expression Omnibus (GEO) database. Difference analysis for MTLE versus control groups under the three time points was conducted to select the differentially expressed genes (DEGs). Time series clustering analysis was used to select the trend genes. Then a series of bioinformatics analyses including functional enrichment analysis, protein–protein interaction (PPI) network and module analyses, and transcription factor (TF) and miRNA prediction were performed. Also, the overall survival analysis and expression of hub genes in LGG were performed using UALCAN from TCGA database. At 6 h, there were 351 upregulated and 80 downregulated DEGs. At 12 h, there were 499 upregulated and 231 downregulated DEGs. Additionally, 532 upregulated and 402 downregulated DEGs were obtained at 24 h. After time series clustering analysis of the DEGs, we obtained 323 uptrend and 248 downtrend genes. We identified 10 key genes with higher degrees, including C3, TIMP1, PENK, CKAP4, etc. Five PPI modules were identified by MCODE. TF analysis predicted four TFs: JUN, STAT3, NR4A2, and Myc. A total of 26,834 miRNA–mRNA pairs were predicted. Moreover, survival analysis of UALCAN suggested that C3, TIMP1, PENK, GNG2, CKAP4, TNC, JUN, STAT3, NR4A2, and Myc can be potential biomarkers for the prognosis of LGG. In summary, DEGs and hub genes were identified in the present study, which provides novel insight into the development of MTLE.
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MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as new potential epigenetic biomarkers. Here, we evaluate the efficacy of six circulating miRNA previously described in the literature as biomarkers for the diagnosis of temporal lobe epilepsy (TLE) and/or as predictive biomarkers to antiepileptic drug response. We measured the differences in serum miRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays in a cohort of 27 patients (14 women and 13 men; mean ± SD age: 43.65 ± 17.07) with TLE compared to 20 healthy controls (HC) matched for sex, age and ethnicity (11 women and 9 men; mean ± SD age: 47.5 ± 9.1). Additionally, patients were classified according to whether they had drug-responsive (n = 17) or drug-resistant (n = 10) TLE. We have investigated any correlations between miRNAs and several electroclinical parameters. Three miRNAs (miR-142, miR-146a, miR-223) were significantly upregulated in patients (expressed as average expression ± SD). In detail, miR-142 expression was 0.40 ± 0.29 vs. 0.16 ± 0.10 in TLE patients compared to HC (t-test, p < 0.01), miR-146a expression was 0.15 ± 0.11 vs. 0.07 ± 0.04 (t-test, p < 0.05), and miR-223 expression was 6.21 ± 3.65 vs. 1.23 ± 0.84 (t-test, p < 0.001). Moreover, results obtained from a logistic regression model showed the good performance of miR-142 and miR-223 in distinguishing drug-sensitive vs. drug-resistant TLE. The results of this pilot study give evidence that miRNAs are suitable targets in TLE and offer the rationale for further confirmation studies in larger epilepsy cohorts.
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Mesial temporal lobe epilepsy (mTLE) is a chronic neurological
disorder characterized by recurrent seizures. Accumulating
evidence of microRNAs´ (miRNAs) potential involvement in
pathogenic mechanisms underlying mTLE is emerging. The function
of microRNAs in chronic mTLE is still poorly understood
partially due to the lack of a clear cut miRNA expression
profile of epileptic foci. In the present study, we performed
the whole miRNome profiling analysis of the human brain tissue
employing novel approach combining next generation sequencing
(NGS) with an advanced qPCR technique, miQPCR. miRNA profiles
of the hippocampus of autopsy controls and mTLE patients were
compared, and analysis revealed differential miRNA expression
of 24 miRNAs consistent with published data as well as several
miRNA species never considered to play a role in epilepsy
before. In conclusion, NGS exposed a group of miRNAs
manifesting invariable expression across all samples out of
which stable expression of four miRNAs was validated by
combined methods for future utilization as negative controls or
normalizers.
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Abstract The identification of biomarkers of the transformation of normal to epileptic tissue would help to stratify patients at risk of epilepsy following brain injury and inform new treatment strategies. MicroRNAs (miRNAs) are an attractive option in this direction. In this study, miRNA microarrays were performed on laser-microdissected hippocampal granule cell layer (GCL) and on plasma, at different time points in the development of pilocarpine-induced epilepsy in the rat: latency, first spontaneous seizure and chronic epileptic phase. Sixty-three miRNAs were differentially expressed in the GCL when considering all time points. Three main clusters were identified that separated the control and chronic phase groups from the latency group and from the first spontaneous seizure group. MiRNAs from rats in the chronic phase were compared to those obtained from the laser-microdissected GCL of epileptic patients, identifying several miRNAs (miR-21-5p, miR-23a-5p, miR-146a-5p and miR-181c-5p) that were up-regulated in both human and rat epileptic tissue. Analysis of plasma samples revealed different levels between control and pilocarpine-treated animals for 27 miRNAs. Two main clusters were identified that segregated controls from all other groups. Those miRNAs that are altered in plasma before the first spontaneous seizure, like miR-9a-3p, may be proposed as putative biomarkers of epileptogenesis.
Pilocarpine
Granule cell
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Epilepsy is one of the most prevalent neurological disorders affecting ~1% of the population. Medial temporal lobe epilepsy (MTLE) is the most frequent type of epilepsy observed in adults who do not respond to pharmacological treatment. The reason for intractability in these patients has not been systematically studied. Further, no markers are available that can predict the subset of patients who will not respond to pharmacotherapy. To identify potential biomarkers of epileptogenicity, we compared the mRNA profiles of surgically resected tissue from seizure zones with non-seizure zones from cases of intractable MTLE. We identified 413 genes that exhibited ≥2-fold change that were statistically significant across these two groups. Several of these differentially expressed genes have not been previously described in the context of MTLE including claudin 11 (CLDN11) and bone morphogenetic protein receptor, type IB (BMPR1B). In addition, we found significant downregulation of a subset of gamma-aminobutyric acid (GABA) associated genes. We also identified molecules such as BACH2 and ADAMTS15, which are already known to be associated with epilepsy. We validated one upregulated molecule, serine/threonine kinase 31 (STK31) and one downregulated molecule, SMARCA4, by immunohistochemical labeling of tissue sections. These molecules need to be further confirmed in large-scale studies to determine their potential use as diagnostic as well as prognostic markers in intractable MTLE.
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