Identificação de microRNAs em epilepsia experimental e desenvolvimento de nanosistema carreador de inibidores de microRNAs para ensaios funcionais

Abstract

Differential gene expression studies coupled with functional assays have been na effective strategy in the identification of molecular pathways underlying pathologies. Considering the regulatory role of miRs, we used this strategy to investigate miRs associated with the epileptogenic process. Objectives: 1. To identify microRNAs differentially expressed in TLE model and 2. To develop a Layered Double Hydroxide (HDL) nanocarrier for miRs inhibitors. Methods: The expression of 662 miRs in hippocampus from rats subjected to Status epilepticus (SE) induced by intrahippocampal injection of pilocarpine was evaluated by microarray hybridization. The miRs 10a-5p, 128a-3p, 196b-5p, 352 and 324-3p were subjected to individual analysis by RT-qPCR at different times after SE (0h, 24h and chronic) and in drugresistant ELT patients. For the development of the LDH nanocarrier (Mg-Al), we used the coprecipitation method. Complexation of the oligonucleotide to HDL was accomplished by vigorous stirring of the solutions. The nanocomplexes obtained were physicochemically characterized as molecular size and zeta potential through the DLS technique; crystallinity through X-ray diffraction; and chemical groups by FTIR spectroscopy. For the in vitro assays, tEND.1 lineage cells were used, which showed significant expression profile of miR-196b-5p and U6snRNA for validation of the system according to previous analyzes. After treatment with HDLs cell viability was assessed by trypan blue exclusion and MTT techniques. The transfection efficiency of the HDL: Oligo nanocomplex was assessed by flow cytometry. Finally, to test the depletion efficiency at miR levels and following validation of the developed system, the levels of miR 196b-5p transcripts and their predicted target genes, Slc9a6 and HMGA2, were evaluated by RT-qPCR. Results: We identified 73 microRNAs that showed significantly different levels of expression in the hippocampus of euthanized rats in the acute phase of epileptogenesis (24 h after SE). Through RTqPCR, were observed the levels of miRs 196b-5p and 352 increase and miR-128a- 3p decrease in the hippocampus of rats submitted to SE. The levels of miR-128a-3β were significantly reduced at all time points compared to naive and miR-352 in the acute and chronic (epileptic rats) period. We observed that the expression levels of miRs-196b-5p and 128a-3p in the hippocampus of epileptic patients presented the same direction of expression compared to those obtained in an animal model. The developed LDH (Mg-Al) nanocarrier presented an average size of 90 d.m and zeta potential of +45 mV, besides standard diffractogram and specific chemical groups for HDL. After complexation, there was a decrease in crystallinity, an increase in particle size and a decrease in zeta potential (mean of 160 d.m., ranging from -15 to -37 mV) indicating effective complexation. The transfection efficiency of the nanocomplex was 93% and absence of toxicity at concentrations up to 100 μg/ml, which shows high biocompatibility. As for the functional assays using the produced nanosystem, significant depletion was observed in transcript levels of miR 196b-5p, 48h after treatment, validating the nanosystem produced here. It has been identified that miRs 196b-5p, 352 and 128-3p are differentially expressed in the ELT model and have developed an efficient tool for functional assays based on miRs depletion. As a perspective, we will investigate the role of mirs 196b-5p in epileptogenesis in animal models using the HDL carrier nanosystem developed here.