Análise da expressão gênica diferencial de micrornas em modelo de epilepsia do lobo temporal

Abstract

Epilepsy is a serious neurological disorder, characterized by recurrent epileptic crisis that affects about 1% of world’s population. Due to the high prevalence and gravity, the temporal lobe’s epilepsy is the most studied. A kind of study in the field of epileptology that had great advances lately is about the investigation of the epileptogenic process, associated with molecular pathways. Studies in animals models and in humans have shown that the epileptogenic process involves a global reorganization of the gene’s expression in the central nervous system. In this context, a raised question is: what are the expression’s regulatory mechanisms of those genes on epileptic tissue? It’s possible that the miRNAs, important molecules in post-transcriptional and translational regulation’s process of the gene expressions, presenting a fundamental role in the determination of a compatible biochemical environment with the epileptogenicity. Our group has investigated this question in an animal model of temporal lobe epilepsy, induced by a pilocarpine injection. In a previous study, by using the approach of hybridization in microarray, we identified 77 microRNAs expressed in different ways in the hippocampus of those animals that were submitted to Status epilepticus (SE) by pilocarpine. In order to start the process of validating these data, we selected 7 microRNAs to evaluate the expression profile during the epileptogenic process. We used Wistar rats injected with pilocarpine (ip) and euthanized immediately and 24 hours after SE, and during the chronic phase (after the establishment of spontaneous recurrent seizures), as controls naive rats were used. The analyzes were conducted by relative expression analysis by RT-qPCR using total RNA purified samples of hippocampus. First, we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. Thereafter, we use these reference genes to analyze the profile of hippocampal expression of the seven microRNAs. The miR-196b showed relative levels of transcripts significantly increased in groups 24 compared to naive animals. The overexpression of miR-352 also presented in 24h group when compared with naïve group, 0h and chronic. The other miRs showed no significant changes in any point of the epileptogenic process. To understand the biological significance of these changes, it is necessary to further study based on functional assays.