Efeito da melatonina na modulação dos miRNAs envolvidos na angiogênese em câncer de mama
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Data
2019-02-18
Autores
Lacerda, Jéssica Zani
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Universidade Estadual Paulista (Unesp)
Resumo
O câncer de mama apresenta alta incidência e alta taxa de mortalidade devido a rápida progressão tumoral e disseminação metastática que ocorrem com o incentivo da angiogênese. MicroRNAs (miRNAs) são pequenas moléculas de RNA mensageiro (RNAm) não codificantes que desempenham papel fundamental na regulação gênica. A desregulação dessas moléculas está relacionada com a iniciação e progressão de diferentes tipos tumorais humanos, incluindo o câncer de mama. Existem fortes evidências de miRNAs atuando como oncogenes e supressores tumorais, regulando o processo de angiogênese, crescimento tumoral e metástase. É neste cenário que a melatonina, hormônio secretado pela glândula pineal, vem ganhando destaque como potencial tratamento contra o câncer de mama por apresentar efeitos oncostáticos, antiangiogênicos, além de atuar na regulação da expressão de miRNAs. Assim, o objetivo deste estudo foi avaliar o potencial valor terapêutico da melatonina na regulação do supressor tumoral miR148a-3p e relação com a progressão tumoral e angiogênese. Inicialmente, células tumorais de mama MDA-MB-231 foram cultivadas nas condições experimentais controle e tratamento com melatonina (1 mM). O PCR Array foi realizado para análise da expressão de 84 miRNAs relacionados com o câncer de mama e, após análise em banco de dados e literatura, o miR-148a-3p foi selecionado para validação. As células MDA-MB-231 foram cultivadas em quatro grupos experimentais: controle, tratamento com melatonina (1 mM), superexpressão do miR-148a-3p e controle do processo de superexpressão e, após, foi realizado PCR em tempo real para análise da expressão gênica do miR-148a-3p e seus alvos Receptor do fator de crescimento semelhante a insulina-tipo 1 (IGF-1R) e Fator de crescimento do endotélio vascular (VEGF). Os níveis proteicos de IGF-1R e VEGF foram quantificados por imunocitoquímica e western blotting. Ensaios de migração e invasão celular foram realizados para avaliar a potencial capacidade das células tumorais sofrerem metástases. Por fim, células MDA-MB-231 foram implantadas no flanco de camundongos Balb-C nude atímicos para desenvolvimento tumoral, sendo divididos em dois grupos experimentais mantidos por 21 dias: controle e melatonina (40 mg/kg). Após a coleta dos tumores, foi realizada a análise da expressão gênica e proteica do miR-148a-3p, IGF-1R e VEGF. A análise da expressão dos miRNAs por PCR array revelou que a melatonina regulou a expressão de 24 miRNAs negativamente e 8 miRNAs positivamente. Além disso, a melatonina aumentou a expressão gênica do miR-148a-3p, diminuiu a expressão gênica e proteica de IGF1R e VEGF, e teve efeito inibitório na sobrevivência, migração e invasão celular. O modelo xenográfico mostrou que a melatonina aumentou a expressão do miR-148a3p e diminuiu a expressão gênica e proteica de IGF-1R e VEGF. Nossos resultados confirmam a ação da melatonina na regulação do miR-148a-3p e processo de angiogênese tumoral, além de desempenhar importante papel na progressão do tumor de mama, sendo crucial para o estabelecimento de novos protocolos terapêuticos.
Breast cancer has a high incidence and high mortality rate due to rapid tumor progression and metastatic dissemination that occur with the encouragement of angiogenesis. MicroRNAs (miRNAs) are small non-coding messenger RNA (mRNA) molecules that play a key role in gene regulation. Deregulation of these molecules is related to the initiation and progression of different human tumor types, including breast cancer. There is strong evidence for miRNAs acting as oncogenes and tumor suppressors, regulating the process of angiogenesis, tumor growth and metastasis. It is in this scenario that melatonin, a hormone secreted by the pineal gland, has been gaining prominence as a potential treatment against breast cancer for having oncostanic and antiangiogenic effects, in addition to regulating the expression of miRNAs. Thus, the objective of this study was to evaluate the potential therapeutic value of melatonin in the regulation of the tumor suppressor miR-148a-3p and its relation to tumor progression and angiogenesis. Initially, MDA-MB-231 breast tumor cells were cultured under the experimental control and treatment conditions with melatonin (1 mM). The PCR Array was performed to analyze the expression of 84 miRNAs related to breast cancer and, after analysis in the database and literature, miR-148a-3p was selected for validation. MDA-MB-231 cells were cultured in four experimental groups: control, treatment with melatonin (1 mM), overexpression of miR-148a-3p and control of the overexpression process, and then real-time PCR was performed for analysis of gene expression of miR-148a-3p and its targets Insulin-like growth factor-1 receptor (IGF-1R) and vascular endothelial growth factor (VEGF). Protein levels of IGF-1R and VEGF were quantified by immunocytochemistry and western blotting. Migration and cell invasion assays were performed to assess the potential ability of tumor cells to undergo metastasis. Finally, MDA-MB-231 cells were implanted on the flank of nude Balb-C mice for tumor development, divided into two experimental groups maintained for 21 days: control and melatonin (40 mg / kg). After collection of the tumors, the analysis of the gene and protein expression of miR148a-3p, IGF-1R and VEGF was performed. Analysis of miRNA expression by PCR array revealed that melatonin regulated expression of 24 negatively miRNAs and 8 miRNAs positively. In addition, melatonin increased miR-148a-3p gene expression, decreased gene and protein expression of IGF-1R and VEGF, and had an inhibitory effect on cell survival, migration, and invasion. The xenographic model showed that melatonin increased the expression of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF. Our results confirm the action of melatonin on the regulation of miR-148a-3p and tumor angiogenesis process, and play an important role in the progression of breast tumor, being crucial for the establishment of new therapeutic protocols.
Breast cancer has a high incidence and high mortality rate due to rapid tumor progression and metastatic dissemination that occur with the encouragement of angiogenesis. MicroRNAs (miRNAs) are small non-coding messenger RNA (mRNA) molecules that play a key role in gene regulation. Deregulation of these molecules is related to the initiation and progression of different human tumor types, including breast cancer. There is strong evidence for miRNAs acting as oncogenes and tumor suppressors, regulating the process of angiogenesis, tumor growth and metastasis. It is in this scenario that melatonin, a hormone secreted by the pineal gland, has been gaining prominence as a potential treatment against breast cancer for having oncostanic and antiangiogenic effects, in addition to regulating the expression of miRNAs. Thus, the objective of this study was to evaluate the potential therapeutic value of melatonin in the regulation of the tumor suppressor miR-148a-3p and its relation to tumor progression and angiogenesis. Initially, MDA-MB-231 breast tumor cells were cultured under the experimental control and treatment conditions with melatonin (1 mM). The PCR Array was performed to analyze the expression of 84 miRNAs related to breast cancer and, after analysis in the database and literature, miR-148a-3p was selected for validation. MDA-MB-231 cells were cultured in four experimental groups: control, treatment with melatonin (1 mM), overexpression of miR-148a-3p and control of the overexpression process, and then real-time PCR was performed for analysis of gene expression of miR-148a-3p and its targets Insulin-like growth factor-1 receptor (IGF-1R) and vascular endothelial growth factor (VEGF). Protein levels of IGF-1R and VEGF were quantified by immunocytochemistry and western blotting. Migration and cell invasion assays were performed to assess the potential ability of tumor cells to undergo metastasis. Finally, MDA-MB-231 cells were implanted on the flank of nude Balb-C mice for tumor development, divided into two experimental groups maintained for 21 days: control and melatonin (40 mg / kg). After collection of the tumors, the analysis of the gene and protein expression of miR148a-3p, IGF-1R and VEGF was performed. Analysis of miRNA expression by PCR array revealed that melatonin regulated expression of 24 negatively miRNAs and 8 miRNAs positively. In addition, melatonin increased miR-148a-3p gene expression, decreased gene and protein expression of IGF-1R and VEGF, and had an inhibitory effect on cell survival, migration, and invasion. The xenographic model showed that melatonin increased the expression of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF. Our results confirm the action of melatonin on the regulation of miR-148a-3p and tumor angiogenesis process, and play an important role in the progression of breast tumor, being crucial for the establishment of new therapeutic protocols.
Descrição
Palavras-chave
Angiogênese, Câncer de mama triplo-negativo, Melatonina, MicroRNA, Pineal, Angiogenesis, Triple-negative breast cancer, Melatonin