Estabelecimento de um modelo celular pró-esteatótico e as particularidades mecanísticas de atuação do microRNA-1914-5p no metabolismo lipídico
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Data
2020-03-13
Autores
Barbosa, Thaís Porto
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Universidade Estadual Paulista (Unesp)
Resumo
As doenças hepáticas estão entre as principais causas de morte no mundo todo, afetando aproximadamente 20% da população. A esteatose hepática é uma condição caracterizada pelo acúmulo de gordura nos hepatócitos e, embora seja uma condição reversível, pode progredir para quadro de esteatohepatite e carcinoma hepatocelular. A falta de tratamentos específicos para a esteatose é um problema atual, e a caracterização de novas moléculas poderá ajudar no desenvolvimento de terapias. Diversos estudos apontam um papel decisivo de microRNAs (miRNAs) no desenvolvimento de patologias hepáticas. Os miRNA são pequenas moléculas que estão envolvidas na regulação de genes importantes no metabolismo lipídico. Mais especificamente, o miR-1914-5p está intimamente relacionado com a biossíntese de ácidos graxos em células hepáticas estreladas, e buscar entender sua atuação funcional no quadro pró-esteatótico celular é o objetivo central dessa proposta. Para isto, um modelo celular pró-esteatótico foi estabelecido no presente trabalho, utilizando-se linhagens celulares hepáticas, tais como LX-2 e HepG2. Ensaios bioquímicos para mensuração de lipídeos intracelulares e níveis de β-oxidação foram realizados. Em células cocultivadas, onde houve um aumento dos níveis endógenos do miR-1914-5p, observou-se um aumento no conteúdo lipídico intracelular, sugerindo um efeito acentuado deste miRNA na produção e acúmulo de lipídeos. Sob outra perspectiva, células cocultivadas onde o miR-1914-5p foi inibido, houve diminuição nos níveis de lipídeos totais e glicose intracelulares, inferindo que este miRNA estaria diminuindo espécies lipídicas que são alteradas no quadro pró-esteatótico. Análises bioquímicas e de expressão gênica foram realizadas em células LX-2. Em células onde há aumento endógeno do miR-1914-5p, houve aumento nos níveis de triglicerídeos e colesterol, além da modulação dos níveis de expressão de diversos genes correlatos à síntese, armazenamento e hidrólise de lipídeos, sugerindo que as células contendo esta condição estariam tentando encontrar uma homeostase lipídica ao liberar AGs para oxidação. Por outro lado, células onde o miR-1914-5p foi inibido, houve diminuição nos níveis de colesterol intracelulares, além deste miRNA ter mostrado interagir com sítios putativos de genes correlatos à síntese de colesterol. A partir dos resultados, a inibição do miR-1914-5p pareceu ter um efeito mais intenso na síntese de lipídeos, uma vez que genes relacionados à via oxidativa não foram modulados. Os resultados obtidos neste trabalho mostraram que o efeito do miR-1914-5p parece variar em linhagens cultivadas isoladamente, tendo a confirmação de que a potencial ação deste miRNA é aparentemente fortalecida quando este é inserido em linhagens cocultivadas.
Liver disease is among the main causes of death worldwide, affecting approximately 20% of the population. Liver steatosis is a condition characterized by the accumulation of fat in hepatocytes and, although it is a reversible condition, it can progress to steatohepatitis and hepatocellular carcinoma. The lack of specific treatments for steatosis is a current problem, and the characterization of new molecules may help in the development of therapies. Several studies point to a decisive role of microRNAs (miRNAs) in the development of liver pathologies. MiRNA are small molecules that are involved in the regulation of important genes in lipid metabolism. More specifically, miR-1914-5p is closely related to the biosynthesis of fatty acids in hepatic stellate cells, and seeking to understand their functional role in the cellular pro-steatotic setting is the central objective of this proposal. For this, a pro-steatotic cell model was established in the present work, using liver cell lines, such as LX-2 and HepG2. Biochemical tests to measure intracellular lipids and β-oxidation levels were performed. In cocultivated cells where there was an increase in endogenous levels of miR-1914-5p, an increase in intracellular lipid content was observed, suggesting a marked effect of this miRNA on lipid production and accumulation. From another perspective, cocultivated cells where miR-1914-5p was inhibited, there was a decrease in the levels of total lipids and intracellular glucose, inferring that this miRNA would be decreasing lipid species that are altered in the pro-steatotic setting. In addition, biochemical and gene expression analyzes were performed on LX-2 cells. In LX-2 cells where there was an endogenous increase in miR-1914-5p, there was an increase in the levels of triglycerides and cholesterol, in addition to the modulation of the expression levels of several genes related to the synthesis, storage and hydrolysis of lipids, suggesting that the cells containing this condition would be trying to find a lipid homeostasis by releasing AGs for oxidation. On the other hand, LX-2 cells where miR-1914-5p was inhibited, there was a decrease in intracellular cholesterol levels, in addition to this miRNA being shown to interact with putative sites of genes related to cholesterol synthesis. From the results, the inhibition of miR-1914-5p in LX-2 cells seemed to have a more intense effect on lipid synthesis, since genes related to the oxidative pathway were not modulated. The results obtained in this project have shown that the effect of miR-1914-5p seems to vary in cell line grown alone, confirming that the potential action of this miRNA is apparently strengthened when it is inserted into cocultured cell line.
Liver disease is among the main causes of death worldwide, affecting approximately 20% of the population. Liver steatosis is a condition characterized by the accumulation of fat in hepatocytes and, although it is a reversible condition, it can progress to steatohepatitis and hepatocellular carcinoma. The lack of specific treatments for steatosis is a current problem, and the characterization of new molecules may help in the development of therapies. Several studies point to a decisive role of microRNAs (miRNAs) in the development of liver pathologies. MiRNA are small molecules that are involved in the regulation of important genes in lipid metabolism. More specifically, miR-1914-5p is closely related to the biosynthesis of fatty acids in hepatic stellate cells, and seeking to understand their functional role in the cellular pro-steatotic setting is the central objective of this proposal. For this, a pro-steatotic cell model was established in the present work, using liver cell lines, such as LX-2 and HepG2. Biochemical tests to measure intracellular lipids and β-oxidation levels were performed. In cocultivated cells where there was an increase in endogenous levels of miR-1914-5p, an increase in intracellular lipid content was observed, suggesting a marked effect of this miRNA on lipid production and accumulation. From another perspective, cocultivated cells where miR-1914-5p was inhibited, there was a decrease in the levels of total lipids and intracellular glucose, inferring that this miRNA would be decreasing lipid species that are altered in the pro-steatotic setting. In addition, biochemical and gene expression analyzes were performed on LX-2 cells. In LX-2 cells where there was an endogenous increase in miR-1914-5p, there was an increase in the levels of triglycerides and cholesterol, in addition to the modulation of the expression levels of several genes related to the synthesis, storage and hydrolysis of lipids, suggesting that the cells containing this condition would be trying to find a lipid homeostasis by releasing AGs for oxidation. On the other hand, LX-2 cells where miR-1914-5p was inhibited, there was a decrease in intracellular cholesterol levels, in addition to this miRNA being shown to interact with putative sites of genes related to cholesterol synthesis. From the results, the inhibition of miR-1914-5p in LX-2 cells seemed to have a more intense effect on lipid synthesis, since genes related to the oxidative pathway were not modulated. The results obtained in this project have shown that the effect of miR-1914-5p seems to vary in cell line grown alone, confirming that the potential action of this miRNA is apparently strengthened when it is inserted into cocultured cell line.
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Palavras-chave
Esteatose hepática, Técnicas de cocultura, Lipídios, Fígado-Doenças, Metabolismo