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MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy - comparison with human epileptic samples

Roncon, Paolo, Soukupovà, Marie, Binaschi, Anna, Falcicchia, Chiara, Zucchini, Silvia, Ferracin, Manuela, Langley, Sarah R., Petretto, Enrico, Johnson, Michael R., Marucci, Gianluca, Michelucci, Roberto, Rubboli, Guido, Simonato, Michele·2015·Scientific Reports
DOI 10.1038/srep14143PMC PMC4585664
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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.

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Figure 1 Cluster analysis of miRNAs differentially expressed in the granule cell layer (GCL) at different time points in the course of pilocarpine-induced epilepsy. Heat map representation of the average expression of the 63 differentially expressed miRNAs in the GCL of epileptic and control animals. Each column represents an individual animal and each row represents one miRNA, as indicated. Colors represent the expression level fold change: higher-red, lower-green. Analysis was performed on differentially expressed miRNAs (FDR > 10%), using the Person correlation; for cluster image preparation an additional step of normalization on gene median across all samples was added.
Figure 1 Cluster analysis of miRNAs differentially expressed in the granule cell layer (GCL) at different time points in the course of pilocarpine-induced epilepsy. Heat map representation of the average expression of the 63 differentially expressed miRNAs in the GCL of epileptic and control animals. Each column represents an individual animal and each row represents one miRNA, as indicated. Colors represent the expression level fold change: higher-red, lower-green. Analysis was performed on differentially expressed miRNAs (FDR > 10%), using the Person correlation; for cluster image preparation an additional step of normalization on gene median across all samples was added.CC BY · PubMed Central
Figure 2 Time course patterns of miRNA expression in the rat granule cell layer (GCL). The boxplots depict the time course of expression for 6 of the miRNAs identified as significant with a false discovery rate (FDR) < 10% in the GCL by using one-way ANOVA. Each boxplot represents 4 animals. These miRNAs were chosen as representative of the different patterns that were observed: up-regulation (miR-21-5p) or down-regulation (miR-222-3p) during latency; up-regulation (miR-181c-5p) or down-regulation (miR-500-3p) in the chronic period; up-regulation (miR-146a-5p) or down-regulation (miR-551b-3p) in the entire course of the disease. The time courses of the other significantly modified miRNAs are shown in Supplementary Figs. S2, S3, S4, S5 and S6. *p < 0.05; **p < 0.01; ***p < 0.001; Tukey’s test.
Figure 2 Time course patterns of miRNA expression in the rat granule cell layer (GCL). The boxplots depict the time course of expression for 6 of the miRNAs identified as significant with a false discovery rate (FDR) < 10% in the GCL by using one-way ANOVA. Each boxplot represents 4 animals. These miRNAs were chosen as representative of the different patterns that were observed: up-regulation (miR-21-5p) or down-regulation (miR-222-3p) during latency; up-regulation (miR-181c-5p) or down-regulation (miR-500-3p) in the chronic period; up-regulation (miR-146a-5p) or down-regulation (miR-551b-3p) in the entire course of the disease. The time courses of the other significantly modified miRNAs are shown in Supplementary Figs. S2, S3, S4, S5 and S6. *p < 0.05; **p < 0.01; ***p < 0.001; Tukey’s test.CC BY · PubMed Central
Figure 3 Network analysis. Predicted gene targets for each miRNA dys-regulated during latency (4 and 8 days after SE), obtained using the mirDB and TargetScan algorithms, were filtered with all miRNAs expressed in the dentate gyrus of rats (database GSE49850). (A) Clusters of miRNAs identified at 10% false discovery rate (FDR) level. (B) Cluster 1. (C) Cluster 3; (D) Cluster 4. (E) Cluster 5. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using webgestalt, p < 0.05.
Figure 3 Network analysis. Predicted gene targets for each miRNA dys-regulated during latency (4 and 8 days after SE), obtained using the mirDB and TargetScan algorithms, were filtered with all miRNAs expressed in the dentate gyrus of rats (database GSE49850). (A) Clusters of miRNAs identified at 10% false discovery rate (FDR) level. (B) Cluster 1. (C) Cluster 3; (D) Cluster 4. (E) Cluster 5. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using webgestalt, p < 0.05.CC BY · PubMed Central
Figure 4 Relative expression of selected miRNAs in the granule cell layer, as detected using qPCR. Five rats per group. *p < 0.05; **p < 0.01; Tukey’s test.
Figure 4 Relative expression of selected miRNAs in the granule cell layer, as detected using qPCR. Five rats per group. *p < 0.05; **p < 0.01; Tukey’s test.CC BY · PubMed Central
Figure 5 Plasma miRNA levels. (A) Heat map representation of the average levels of the 27 differentially expressed miRNAs in the plasma of epileptic and control animals. Each column represents an individual animal and each row represents one miRNA, as indicated. Colors represent the expression level fold change: higher-red, lower-green. Analysis was performed on differentially represented miRNAs using the Person correlation. (B) The boxplots depict the time course for 4 of the miRNAs identified as significant by using one-way ANOVA. Each boxplot represents 5 animals. These miRNAs were chosen as representative of the different patterns that were observed: up-regulation (miR-9a-5p) or down-regulation (miR-598-5p) during latency, down-regulation in the late latency - first spontaneous seizure period (miR-381-3p) and down regulation in the chronic stage (miR-142-5p). (C) Relative levels of miR-9a-5p and miR-142-3p, as detected using qPCR. Five rats per group. *p < 0.05; **p < 0.01; ***p < 0.001; Tukey’s test.
Figure 5 Plasma miRNA levels. (A) Heat map representation of the average levels of the 27 differentially expressed miRNAs in the plasma of epileptic and control animals. Each column represents an individual animal and each row represents one miRNA, as indicated. Colors represent the expression level fold change: higher-red, lower-green. Analysis was performed on differentially represented miRNAs using the Person correlation. (B) The boxplots depict the time course for 4 of the miRNAs identified as significant by using one-way ANOVA. Each boxplot represents 5 animals. These miRNAs were chosen as representative of the different patterns that were observed: up-regulation (miR-9a-5p) or down-regulation (miR-598-5p) during latency, down-regulation in the late latency - first spontaneous seizure period (miR-381-3p) and down regulation in the chronic stage (miR-142-5p). (C) Relative levels of miR-9a-5p and miR-142-3p, as detected using qPCR. Five rats per group. *p < 0.05; **p < 0.01; ***p < 0.001; Tukey’s test.CC BY · PubMed Central
Figure 6 Laser microdissection of the rat granule cell layer. Representative hematoxylin stained hippocampal section prepared from a pilocarpine treated rat (level: dentate gyrus of the hippocampus). (A) Intact section before laser microdissection. (B) Identification of the region of interest, the granular layer (black line). (C) The section after the laser microdissection, without the granular layer. Horizontal bar = 320 μm.
Figure 6 Laser microdissection of the rat granule cell layer. Representative hematoxylin stained hippocampal section prepared from a pilocarpine treated rat (level: dentate gyrus of the hippocampus). (A) Intact section before laser microdissection. (B) Identification of the region of interest, the granular layer (black line). (C) The section after the laser microdissection, without the granular layer. Horizontal bar = 320 μm.CC BY · PubMed Central

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Metadata

Authors
Roncon, P., Soukupovà, M., Binaschi, A., Falcicchia, C., Zucchini, S., Ferracin, M., Langley, S. R., Petretto, E., Johnson, M. R., Marucci, G., Michelucci, R., Rubboli, G., & Simonato, M.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2015-09-18
Volume
5
Issue
1
ISSN
2045-2322
DOI
10.1038/srep14143
PMC
PMC4585664
Cited by
98
References
1
Language
en
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