NormFinder analysis of expression stability of candidate reference genes for microRNA qRT-PCR analysis in the systemic PILO-model samples.

<p>Ranking of candidate reference genes based on stability values calculated by NormFinder software.</p

Levels of five non-coding RNAs candidate reference genes for microRNA qRT-PCR analysis in the hippocampus of the systemic PILO-injected and control rats.

<p>Values are given in the form of RT-qPCR threshold cycle numbers (Ct values), mean SD (n = 6). ANOVA, * , ** , *** .</p

Selection of the most suitable reference genes for microRNA qRT-PCR analysis in the systemic PILO-model samples using geNorm analysis.

<p>A) Expression stability measurements (M) for the five reference genes analyzed. The x-axis from left to right indicates the ranking of the genes according to their expression stability; lower M values indicate higher expression stability. B) Determination of the optimal number of reference genes for normalization was conducted. The software calculates the normalization factor from at least two genes at which the variable V defines the pair-wise variation between two sequential normalization factors.</p

Relative quantities of miR-146a in the hippocampus of the systemic PILO- injected rats upon different normalization approaches.

<p>qRT-PCR data were normalized by single reference gene and best combination derived by geNorm or NormFinder analysis (mean SD, n = 6). The diagram shows mean levels of miR-146a transcripts in naive animals, epileptogenesis (0 h and 24 h) and chronic period. ANOVA, * , ** , *** .</p

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis

<div><p>The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naïve group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis.</p></div

RT-qPCR validation of microarray results.

<p>miRs 196b-5p and 352 were significantly increased and miR-128a-3p was significantly reduced in 24h post-SE hippocampus compared with control group. miRs 10a-5p and 324-3p did not show statistically significant differences. (values are mean±SEM, n = 5-6 in each group, *p < 0.05, Unpaired t test).</p

Expression patterns of miR-128a-3p, miR-196b-5p and miR-352 during S-PILO-SE induced epileptogenesis.

<p>RT-qPCR measurements of the relative miRs levels (hippocampus) in three different time points after SE-induction (values are mean±SEM, n = 5-6 in each group, *p < 0.05, Bonferroni’s Multiple Comparison Test.</p

GO Pathway significantly overrepresented among the miR-196b-5p validated targets.

<p>GO Pathway significantly overrepresented among the miR-196b-5p validated targets.</p

GO Pathway significantly overrepresented among the miR-128a-3p validated targets.

<p>GO Pathway significantly overrepresented among the miR-128a-3p validated targets.</p

Relative expression of miR-128a-3p and miR-196b-5p in hippocampi from epileptic and non-epileptic humans.

<p>RT-qPCR analysis (values are meanSEM, biopsy specimens from TLE-HS patients (n = 10-14) and non-epileptic autopsy samples (n = 4), Mann Whitney test).</p