Validação funcional de genes-alvo de microRNAs em carcinoma hepatocelular
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2020-08-26
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Universidade Estadual Paulista (Unesp)
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Introdução: miRNAs são pequenos RNAs não codificadores de proteínas que regulam a expressão gênica associada a diversos mecanismos biológicos. Os miRNAs têm sido indicados como potenciais biomarcadores para o diagnóstico, prognóstico e tratamento de diversos tipos de câncer, incluindo o carcinoma hepatocelular (CHC). CHC é a quarta causa mais comum de morte por câncer e é frequentemente associado com mau prognóstico, agressividade, resistente a medicamentos e diagnostico avançado. Objetivo: Identificar e validar experimentalmente a interação entre miRNA/genes-alvo relacionados com o processo de tumorigênese do CHC e contribuir para a identificação de vias moleculares relevantes para o desenvolvimento tumoral. Além disso, verificar o efeito da alteração da expressão do miRNA escolhido sobre o potencial de proliferação, migração e invasão celular nas linhagens de CHC. Material e Métodos: A seleção do miRNA foi feita a partir de dados anteriores do grupo e análises de bioinformática. A identificação dos genes-alvo foi realizada por meio de ferramentas de predição computacional (mirDIP e miRTarBase). As linhagens celulares de câncer de CHC (Hep 3B e PCL/PRF/5) foram transfectadas com o mimético do miRNA selecionado, ou o seu controle negativo, a fim de avaliarmos a expressão dos genes-alvo (RT-qPCR). Após a transfecção, foi avaliado o potencial de proliferação celular (MTS), migração (wound healing) e invasão (ensaio transwell) das linhagens. Ensaio da Luciferase foi utilizado para confirmar a ligação do miRNA ao gene-alvo. Resultados: O miR-451a foi selecionado para a validação funcional por apresentar consistentemente diminuição de expressão em CHC de três bancos de dados (tumores primários de pacientes, meta-análise e TCGA). Análise in silico revelou 12 possíveis genes candidatos regulados pelo miR-451a relevantes na tumorigênese e ainda não validados experimentalmente: FMNL3, BUB1B, SPC25, KIF2A, PLEKHG2, BAK1, HMGB2, MEF2D, CDKN2B, HELLS, RRAGD, CSE1L. Nas duas linhagens celulares, o aumento da expressão do miR-451a após transfecção com miRNA mimético resultou na diminuição significativa (p<0,05) da expressão do gene SPC25. O ensaio de luciferase demonstrou a ligação do microRNA ao gene SPC2, confirmando que os gene SPC25 é alvo direto do miR-451a. Além disso, a restauração da expressão do miR-451a levou à diminuição de proliferação, migração e invasão nas linhagens celulares Hep 3B e PCL/PRF/5. Conclusão: O gene SPC25 foi identificado como um novo alvo do miR-451a e associado a CHC. Além disso, nossos achados indicam que o miR-451a está relacionado à tumorigênese e progressão do CHC, sendo sugerido como um potencial alvo para a terapia do CHC.
Introduction: MicroRNAS (miRNAs) are small non-coding RNAs that and regulate the expression of genes associated with several biological mechanisms. miRNAs have been indicated as potential biomarkers for the diagnosis, prognosis and treatment of several types of cancer, including hepatocellular carcinoma (HCC). HCC is the fourth most common cause of cancer death and is often associated with poor prognosis, aggression, drug resistance and advanced diagnosis. Aim: To identify and experimentally validate the interaction between miRNA/target genes related to the HCC tumorigenesis process and contribute to the identification of molecular pathways relevant to tumor development. In addition, to verify the effect of changing the expression of the chosen miRNA in the cell proliferation, migration and invasion of the liver cancer cell lines. Material and Methods: The selection of miRNA was performed based on the analysis of previous data of our group and bioinformatic analyses. The identification of the target genes was performed using computational prediction tools (mirDIP and miRTarBase). The HCC cell lines (Hep 3B and PCL/PRF/5) were transfected with the selected miRNA mimic or negative control, in order to evaluate the expression of target genes (RT-qPCR). After transfection, the potential of cell proliferation (MTS), migration (wound healing) and invasion (transwell assay) of the cell lines was evaluated. Luciferase assay was used to confirm miRNA binding to the target gene. Results: miR-451a was selected for functional validation by being consistently detected as underexpressed in HCC from three databases (primary patient tumors, meta-analysis and TCGA). In silico analysis revealed 12 putative candidate genes regulated by miR-451a, relevant in HCC tumorigenesis and not yet experimentally validated: FMNL3, BUB1B, SPC25, KIF2A, PLEKHG2, BAK1, HMGB2, MEF2D, CDKN2B, HELLS, RRAGD and CSE1L. In both cell lines, the increase in miR-451a expression by transfection of the mimic miRNA resulted in a significant decrease (p <0.05) of SPC25 gene expression. The luciferase assay demonstrated the binding of the microRNA to the SPC25 gene, confirming that the SPC25 gene is a direct target of miR-451a. Furthermore, the restoration of miR-451a expression led to decrease in cell proliferation, migration and invasion of the Hep 3B and PCL/PRF/5 cell lines. Conclusion: The SPC25 gene was identified as a new target of miR-451a. Moreover, our findings indicate that miR-451a is related to HCC tumorigenesis and progression, being suggested as a potential target for HCC therapy.
Introduction: MicroRNAS (miRNAs) are small non-coding RNAs that and regulate the expression of genes associated with several biological mechanisms. miRNAs have been indicated as potential biomarkers for the diagnosis, prognosis and treatment of several types of cancer, including hepatocellular carcinoma (HCC). HCC is the fourth most common cause of cancer death and is often associated with poor prognosis, aggression, drug resistance and advanced diagnosis. Aim: To identify and experimentally validate the interaction between miRNA/target genes related to the HCC tumorigenesis process and contribute to the identification of molecular pathways relevant to tumor development. In addition, to verify the effect of changing the expression of the chosen miRNA in the cell proliferation, migration and invasion of the liver cancer cell lines. Material and Methods: The selection of miRNA was performed based on the analysis of previous data of our group and bioinformatic analyses. The identification of the target genes was performed using computational prediction tools (mirDIP and miRTarBase). The HCC cell lines (Hep 3B and PCL/PRF/5) were transfected with the selected miRNA mimic or negative control, in order to evaluate the expression of target genes (RT-qPCR). After transfection, the potential of cell proliferation (MTS), migration (wound healing) and invasion (transwell assay) of the cell lines was evaluated. Luciferase assay was used to confirm miRNA binding to the target gene. Results: miR-451a was selected for functional validation by being consistently detected as underexpressed in HCC from three databases (primary patient tumors, meta-analysis and TCGA). In silico analysis revealed 12 putative candidate genes regulated by miR-451a, relevant in HCC tumorigenesis and not yet experimentally validated: FMNL3, BUB1B, SPC25, KIF2A, PLEKHG2, BAK1, HMGB2, MEF2D, CDKN2B, HELLS, RRAGD and CSE1L. In both cell lines, the increase in miR-451a expression by transfection of the mimic miRNA resulted in a significant decrease (p <0.05) of SPC25 gene expression. The luciferase assay demonstrated the binding of the microRNA to the SPC25 gene, confirming that the SPC25 gene is a direct target of miR-451a. Furthermore, the restoration of miR-451a expression led to decrease in cell proliferation, migration and invasion of the Hep 3B and PCL/PRF/5 cell lines. Conclusion: The SPC25 gene was identified as a new target of miR-451a. Moreover, our findings indicate that miR-451a is related to HCC tumorigenesis and progression, being suggested as a potential target for HCC therapy.
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Carcinoma hepatocelular, microRNAs, miR-451a, Genes-alvo, Vias moleculares, Linhagens tumorais, Validação funcional, Hepatocellular carcinoma, Target genes, Molecular pathways, Tumor cell lines, Functional validation