Análise funcional do microRNA-1914-5p no metabolismo de lipídeos de células hepáticas tumorais
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Data
2019-02-25
Autores
Silveira, Marina Bonfogo da [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O carcinoma hepatocelular ou hepatocarcinoma é um dos tipos mais comuns de câncer no mundo e o com maior taxa de mortalidade devido ao seu diagnóstico tardio. Novas terapias envolvendo o controle da síntese de ácidos graxos é uma potencial estratégia contra a tumorigênese e a progressão tumoral, visto que alterações no metabolismo de lipídeos podem limitar a proliferação de células cancerígenas, assim como uma dieta rica em gorduras pode favorecer doenças que acometem o fígado e até estimular a metástase de alguns cânceres. Pequenas moléculas, como os microRNAs, podem ser usadas como controladoras desse metabolismo e possibilitam o desenvolvimento de novas estratégias terapêuticas. Assim sendo, esse estudo avaliou os efeitos moleculares e celulares do miR-1914-5p em culturas celulares hepáticas, Hep G2 e SK-HEP-1, no que tange o seu papel no metabolismo de lipídeos e a consequência das alterações nessa via para a migração das células. Para isso, foram realizados ensaios de PCR quantitativa e Western Blot para moléculas correlatas ao metabolismo lipídeo, marcação de ácidos graxos por microscopia de luz e fluorescência, além da quantificação desses por cromatografia gasosa acoplada à um espectometro de massa. Também foram realizados ensaios de análise de proliferação e migração celular, como o ensaio da ferida e da gota de agarose. Os resultados mostraram que altos níveis do miR-1914-5p causaram regulação da expressão gênica e altos níveis proteicos da Malonil-CoA Descaboxilase e da Ácido Graxo Sintase. Por consequência, um número elevado de gotículas lipídicas e aumento de ácidos graxos foram observados. Esses resultados combinados com a alta expressão de Lipina 1 sugerem correlação do miR-1914-5p e o acúmulo de gordura em células de hepatocarcinoma. Por outro lado, a inibição do miR-1914-5p parece ter efeito reverso, causando possível ativação da via de β-oxidação caracterizado pela ausência de gotículas lipídicas e alta expressão de Acetil-CoA Carboxilase β. Também foi observado um possível estresse metabólico perante a inibição miR-1914-5p. Esses dados sugerem o potencial modulatório do miR-1914-5p sobre o metabolismo de lipídeos em câncer de fígado.
Hepatocellular carcinoma or hepatocarcinoma is one of the most common types of cancer in the world and the one with the highest mortality rate due to its late diagnosis. New therapies involving the control of fatty acid synthesis is a potential strategy against tumorigenesis and tumor progression, since alterations in lipid metabolism may limit the proliferation of cancer cells, as a diet rich in fats may favor diseases that affect the liver and even stimulates the metastasis of some cancers. Small molecules, such as microRNAs, can be used as controllers of this metabolism and enable the development of new therapeutic strategies. Thus, this study evaluated the molecular and cellular effects of miR-1914-5p on hepatic cell cultures, Hep G2 and SK-HEP-1, as regards its role in lipid metabolism and the consequence of alterations in this pathway to cell migration. For this, quantitative PCR and Western Blot assays were performed for molecules correlated to lipid metabolism, labeling of fatty acids by light microscopy and fluorescence, besides the quantification of these by Gas Chromatography coupled to a Mass Spectrometer. Cell proliferation and migration assays were also performed, such as the Wound Healing and Agarose Assay. The results showed that high levels of miR1914-5p caused regulation of gene expression and high protein levels of Malonyl-CoA Decaboxylase and Fatty Acid Synthase. Consequently, a high number of lipid droplets and increased fatty acids were observed. These results combined with the high expression of Lipin 1 suggest correlation of miR-1914-5p and accumulation of fat in hepatocarcinoma cells. On the other hand, inhibition of miR-1914-5p seems to have a reverse effect, causing possible activation of the β-oxidation pathway characterized by the absence of lipid droplets and high expression of Acetyl-CoA Carboxylase β. Potential metabolic stress was also observed against miR-1914-5p inhibition. These data suggest the modulatory potential of miR-1914-5p on lipid metabolism in liver cancer.
Hepatocellular carcinoma or hepatocarcinoma is one of the most common types of cancer in the world and the one with the highest mortality rate due to its late diagnosis. New therapies involving the control of fatty acid synthesis is a potential strategy against tumorigenesis and tumor progression, since alterations in lipid metabolism may limit the proliferation of cancer cells, as a diet rich in fats may favor diseases that affect the liver and even stimulates the metastasis of some cancers. Small molecules, such as microRNAs, can be used as controllers of this metabolism and enable the development of new therapeutic strategies. Thus, this study evaluated the molecular and cellular effects of miR-1914-5p on hepatic cell cultures, Hep G2 and SK-HEP-1, as regards its role in lipid metabolism and the consequence of alterations in this pathway to cell migration. For this, quantitative PCR and Western Blot assays were performed for molecules correlated to lipid metabolism, labeling of fatty acids by light microscopy and fluorescence, besides the quantification of these by Gas Chromatography coupled to a Mass Spectrometer. Cell proliferation and migration assays were also performed, such as the Wound Healing and Agarose Assay. The results showed that high levels of miR1914-5p caused regulation of gene expression and high protein levels of Malonyl-CoA Decaboxylase and Fatty Acid Synthase. Consequently, a high number of lipid droplets and increased fatty acids were observed. These results combined with the high expression of Lipin 1 suggest correlation of miR-1914-5p and accumulation of fat in hepatocarcinoma cells. On the other hand, inhibition of miR-1914-5p seems to have a reverse effect, causing possible activation of the β-oxidation pathway characterized by the absence of lipid droplets and high expression of Acetyl-CoA Carboxylase β. Potential metabolic stress was also observed against miR-1914-5p inhibition. These data suggest the modulatory potential of miR-1914-5p on lipid metabolism in liver cancer.
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Palavras-chave
miRNA hsa-miR-1914-5p, Metabolismo de lipídeos, Lipid metabolism, Hepatocarcinoma