Busca por miRNAs provenientes de exossomos relevantes para epileptogênese
Data
2022
Autores
Rueda, Beatriz Enguidanos Villena [UNIFESP]
Orientadores
Belangero, Síntia Iole Nogueira [UNIFESP]
Tipo
Trabalho de conclusão de curso de graduação
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O Traumatismo Cranioencefálico (TCE) é altamente frequente no Brasil, ocorrendo mais de 150 mil casos por ano, e é um grande fator de risco para o desenvolvimento de epilepsia pós-traumática (EPT). Contudo, o tratamento de EPT derivado de TCE ainda é pouco eficaz. Portanto, é necessário investigar novas abordagens, como o uso do biperideno, um medicamento anticolinérgico. MicroRNAs (miRNAs) provenientes de vesículas extracelulares (VEs) podem auxiliar na compreensão das bases biológicas da EPT, uma vez que regulam a expressão gênica e podem ser detectados perifericamente. As VEs são liberadas por vários tipos celulares, como células neurais, e são capazes de atravessar a barreira hematoencefálica. O principal objetivo desse estudo foi identificar padrões de alterações nos níveis de miRNAs carreados por VEs após TCE, que possam estar relacionados com o desenvolvimento de EPT e/ou que estejam relacionados com o tratamento com biperideno. Os objetivos específicos, foram: a) encontrar miRNAs associados à EPT, ao comparar o grupo dos indivíduos que desenvolveram com o dos que não desenvolveram a EPT; b) buscar miRNAs relacionados ao tratamento, ao comparar o grupo dos indivíduos que tomaram placebo com o dos que receberam biperideno; e c) identificar os genes alvo dos miRNAs diferencialmente expressos e as suas vias biológicas enriquecidas na epileptogênese e no tratamento com biperideno. A coleta dos participantes do estudo fez parte de um projeto temático que incluiu um ensaio clínico duplo-cego e randomizado. Foram incluídos 37 indivíduos que sofreram TCE, sendo que a coleta de sangue foi realizada após 10 dias do trauma e do início do tratamento (biperideno ou placebo). Desses 37, 18 receberam biperideno e 19 receberam placebo, e ao todo cinco indivíduos desenvolveram EPT. Primeiramente, foi realizado o isolamento de VEs provenientes do soro, sendo uma parte utilizada para a sua caracterização, de acordo com o seu tamanho e a presença de proteínas de membrana. A outra parte foi utilizada para a extração de miRNAs seguido do preparo de bibliotecas para o sequenciamento de nova geração do miRNoma (conjunto de miRNAs). A análise de expressão diferencial desses miRNAs foi realizada com o auxílio de ferramentas de bioinformática. Em relação aos miRNAs associados com o tratamento, o miR-9-5p (p ajustado < 0,05) apresentou uma expressão diminuída em pacientes que tomaram biperideno. A análise de vias para os alvos desse miRNA mostrou um enriquecimento para a via de ciclo de vesículas sinápticas. O miRNA miR-17-5p, que encontramos com a expressão aumentada em pacientes que tomaram biperideno (p < 0,01), tem como alvos genes das vias de morfogênese dendrítica, regulação da neurogênese e desenvolvimento de axônios. Na análise de miRNAs associados a EPT, embora nenhum tenha sido associado após a correção para múltiplas comparações, destacamos o miR-144-3p, que foi encontrado com uma expressão aumentada em pacientes que desenvolveram EPT (p < 0,01). Os alvos desse miRNA estão enriquecidos para as vias de regulação do desenvolvimento da projeção neuronal, axonogênese e regulação da organização sináptica. Os resultados das vias biológicas encontradas como enriquecidas para os alvos desses três miRNAs diferencialmente expressos corroboram estudos da literatura, sendo esses miRNAs associados com processos neuroprotetores e/ou neurodegenerativos. Dessa forma, concluímos que miRNAs expressos em VEs podem ser potenciais marcadores de risco para a EPT, bem como interagir com o biperideno na prevenção dela, auxiliando no melhor prognóstico de indivíduos que sofreram TCE.
Traumatic brain injury (TBI) is highly frequent in Brazil, occurring more than 150,000 cases per year, and is a major risk factor for the development of post-traumatic epilepsy (PTE). However, treatment for PTE derived from TBI is still ineffective. Therefore, the investigation of new approaches is necessary, such as use of biperiden, an anticholinergic drug. MicroRNAs (miRNAs) obtained from extracellular vesicles (EVs) can help to understand PTE’s biological basis, as they regulate gene expression and can be detected peripherally. EVs are released by multiple cell types, such as neural cells, and are able to cross the blood-brain barrier. The main objective of this study is to identify changes in miRNAs levels carried by EVs after TBI, which may be related to the PTE’s development and/or are related to the treatment with biperiden. As specific objectives, we intend to: a) find miRNAs associated with PTE, comparing the groups that developed or did not develop PTE; b) identify miRNAs related to the treatment, when comparing placebo and biperiden-treated groups; c) associate changes in miRNAs levels with clinical variables of the patients, such as, severity and lesion location; and d) identify the target genes of the differentially expressed miRNA and their enriched biological pathways associated with epileptogenesis and treatment with biperiden. The selection of study participants was carried out and was part of a thematic project that included a double-blind and randomized clinical trial. As a part of this project, 37 TBI patients were included. Blood collection of these patients was performed 10 days after the trauma and the beginning of treatment (biperiden or placebo). EVs were isolated from blood serum, part of which was used for characterization of said vesicles, according to their size and the presence of membrane proteins. The remaining EVs were used for the extraction of miRNAs, which were used for next generation sequencing of the miRNoma (set of miRNAs). The miRNAs differential expression analysis was performed using bioinformatics tools. Regarding treatment-associated miRNAs, miR-9-5p (adjusted p < 0.05) showed a decreased expression in patients taking biperiden. Pathway analysis for this miRNA targets showed an enrichment for the synaptic defeat cycle pathway. The miRNA miR-17-5p, which we found with increased expression in patients who took biperiden (p < 0.01), targets genes in dendritic morphogenesis pathways, regulation of neurogenesis and axonal development. In the analysis of miRNAs associated with EPT, although none were associated after correcting for multiple comparisons, we highlighted miR-144-3p, which was found with an increased expression in patients who developed EPT (p < 0.01). The targets of this miRNA are enriched for pathways regulating the development of neuronal projection, axonogenesis and regulation of synaptic organization. The results of the biological pathways found to be enriched for the targets of these three differentially expressed miRNAs corroborate studies in the literature, these miRNAs being associated with neuroprotective and/or neurodegenerative processes. Thus, we conclude that miRNAs expressed in EVs may be potential risk markers for EPT, as well as interact with biperiden in its prevention, helping to improve the prognosis of individuals who have suffered TBI.
Traumatic brain injury (TBI) is highly frequent in Brazil, occurring more than 150,000 cases per year, and is a major risk factor for the development of post-traumatic epilepsy (PTE). However, treatment for PTE derived from TBI is still ineffective. Therefore, the investigation of new approaches is necessary, such as use of biperiden, an anticholinergic drug. MicroRNAs (miRNAs) obtained from extracellular vesicles (EVs) can help to understand PTE’s biological basis, as they regulate gene expression and can be detected peripherally. EVs are released by multiple cell types, such as neural cells, and are able to cross the blood-brain barrier. The main objective of this study is to identify changes in miRNAs levels carried by EVs after TBI, which may be related to the PTE’s development and/or are related to the treatment with biperiden. As specific objectives, we intend to: a) find miRNAs associated with PTE, comparing the groups that developed or did not develop PTE; b) identify miRNAs related to the treatment, when comparing placebo and biperiden-treated groups; c) associate changes in miRNAs levels with clinical variables of the patients, such as, severity and lesion location; and d) identify the target genes of the differentially expressed miRNA and their enriched biological pathways associated with epileptogenesis and treatment with biperiden. The selection of study participants was carried out and was part of a thematic project that included a double-blind and randomized clinical trial. As a part of this project, 37 TBI patients were included. Blood collection of these patients was performed 10 days after the trauma and the beginning of treatment (biperiden or placebo). EVs were isolated from blood serum, part of which was used for characterization of said vesicles, according to their size and the presence of membrane proteins. The remaining EVs were used for the extraction of miRNAs, which were used for next generation sequencing of the miRNoma (set of miRNAs). The miRNAs differential expression analysis was performed using bioinformatics tools. Regarding treatment-associated miRNAs, miR-9-5p (adjusted p < 0.05) showed a decreased expression in patients taking biperiden. Pathway analysis for this miRNA targets showed an enrichment for the synaptic defeat cycle pathway. The miRNA miR-17-5p, which we found with increased expression in patients who took biperiden (p < 0.01), targets genes in dendritic morphogenesis pathways, regulation of neurogenesis and axonal development. In the analysis of miRNAs associated with EPT, although none were associated after correcting for multiple comparisons, we highlighted miR-144-3p, which was found with an increased expression in patients who developed EPT (p < 0.01). The targets of this miRNA are enriched for pathways regulating the development of neuronal projection, axonogenesis and regulation of synaptic organization. The results of the biological pathways found to be enriched for the targets of these three differentially expressed miRNAs corroborate studies in the literature, these miRNAs being associated with neuroprotective and/or neurodegenerative processes. Thus, we conclude that miRNAs expressed in EVs may be potential risk markers for EPT, as well as interact with biperiden in its prevention, helping to improve the prognosis of individuals who have suffered TBI.
Descrição
Citação
VILLENA RUEDA, B E. Busca por miRNAs provenientes de exossomos relevantes para epileptogênese. São Paulo, 2022. 53 f. Trabalho de conclusão de curso (Graduação em Biomedicina) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2022.