Genes e epilepsia II: expressão gênica diferencial

Romcy-Pereira, Rodrigo Neves; Gitaí, Daniel Leite Góes; Gitaí, Lívia Leite Góes; Leite, João Pereira; García-Cairasco, Norberto; Paçó-Larson, Maria Luísa

doi:10.1590/s0104-42302008000500022

Cited by 6 publications

(3 citation statements)

References 48 publications

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“…The pathogenesis of MTLE involves structural and cellular reorganization of the hippocampal formation, including neuron loss, neurogenesis, gliosis, axonal damage or sprouting, dendritic plasticity, inflammation and reorganization of the extracellular matrix [ 1 , 2 ]. The molecular mechanisms underlying these processes have been widely investigated by differential gene expression approaches [ 3 , 4 ]. Combining different terms, such as ‘‘gene expression”, ‘‘pilocarpine”, ‘‘epilepsy” and ‘‘PCR”, we performed a PubMed search for articles published from January 1, 2005 to January 1, 2015 and got 57 available articles that evaluated gene expression changes by RT-PCR in the PILO-model.…”

Section: Introductionmentioning

confidence: 99%

Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

et al. 2015

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The molecular mechanisms underlying epileptogenesis have been widely investigated by differential gene expression approach, especially RT-qPCR methodology. However, controversial findings highlight the occurrence of unpredictable sources of variance in the experimental designs. Here, we investigated if diurnal rhythms of transcript’s levels may impact on differential gene expression analysis in hippocampus of rats with experimental epilepsy. For this, we have selected six core clock genes (Per1, Per3, Bmal1, Clock, Cry1 and Cry2), whose rhythmic expression pattern in hippocampus had been previously reported. Initially, we identified Tubb2a/Rplp1 and Tubb2a/Ppia as suitable normalizers for circadian studies in hippocampus of rats maintained to 12:12 hour light:dark (LD) cycle. Next, we confirmed the temporal profiling of Per1, Per3, Bmal1, Cry1 and Cry2 mRNA levels in the hippocampus of naive rats by both Acrophase and CircWave statistical tests for circadian analysis. Finally, we showed that temporal differences of sampling can change experimental results for Per1, Per3, Bmal1, Cry1 and Cry2, but not for Clock, which was consistently decreased in rats with epilepsy in all comparison to the naive group. In conclusion, our study demonstrates it is mandatory to consider diurnal oscillations, in order to avoid erroneous conclusions in gene expression analysis in hippocampus of rats with epilepsy. Investigators, therefore, should be aware that genes with circadian expression could be out of phase in different animals of experimental and control groups. Moreover, our results indicate that a sub-expression of Clock may be involved in epileptogenicity, although the functional significance of this remains to be investigated.

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Section: Introductionmentioning

confidence: 99%

Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

et al. 2015

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“…An approach that has been widely used is the analysis of differential gene expression in the affected tissue [1]–[4]. Quantitative real-time PCR (RT-qPCR) is currently the gold standard for the quantification of steady-state mRNA levels due to its accuracy and sensitivity [5]–[7].…”

Section: Introductionmentioning

confidence: 99%

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

et al. 2013

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It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

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“…These changes include neurodegeneration, neurogenesis, gliosis, axonal damage or sprouting, dendritic plasticity, and inflammation in hippocampus and other limbic structures [ 5 – 10 ]. Although our understanding of epileptogenesis is incomplete, previous research indicates that it is associated with a network-wide reorganization of gene expression in the affected brain [ 11 – 20 ]. Therefore, uncovering the specific factors that lead to the dysregulation of several genes may provide important insights into the epileptogenic process.…”

Section: Introductionmentioning

confidence: 99%

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis

et al. 2016

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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.

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Genes e epilepsia II: expressão gênica diferencial

Cited by 6 publications

References 48 publications

Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis

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