RESUMO
BACKGROUND: Eukaryotic translation elongation factor 1A2 (eEF1A2) is a known proto-oncogene. We proposed that stimulation of the eEF1A2 expression in cancer tissues is caused by the loss of miRNA-mediated control. METHODS: Impact of miRNAs on eEF1A2 at the mRNA and protein levels was examined by qPCR and western blot, respectively. Dual-luciferase assay was applied to examine the influence of miRNAs on 3'-UTR of EEF1A2. To detect miRNA-binding sites, mutations into the 3'-UTR of EEF1A2 mRNA were introduced by the overlap extension PCR. RESULTS: miR-663 and miR-744 inhibited the expression of luciferase gene attached to the 3'-UTR of EEF1A2 up to 20% and 50%, respectively. In MCF7 cells, overexpression of miR-663 and miR-744 reduced the EEF1A2 mRNA level by 30% and 50%. Analogous effects were also observed at the eEF1A2 protein level. In resveratrol-treated MCF7 cells the upregulation of mir-663 and mir-744 was accompanied by downregulation of EEF1A2 mRNA. Both miRNAs were able to inhibit the proliferation of MCF7 cells. CONCLUSION: miR-663 and miR-744 mediate inhibition of the proto-oncogene eEF1A2 expression that results in retardation of the MCF7 cancer cells proliferation. Antitumour effect of resveratrol may include stimulation of the miR-663 and miR-744 expression.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/terapia , MicroRNAs/administração & dosagem , Fator 1 de Elongação de Peptídeos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Processos de Crescimento Celular/genética , Movimento Celular/genética , Regulação para Baixo , Feminino , Humanos , Células MCF-7 , MicroRNAs/genética , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Fator 1 de Elongação de Peptídeos/biossíntese , Proto-Oncogene Mas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Resveratrol , Estilbenos/farmacologia , TransfecçãoRESUMO
TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial-mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation and EMT, we found TWIST1 to be upregulated during EMT and downregulated early in carcinogenesis. The TWIST1 3'UTR contains putative regulatory elements, including miRNA target sites and two cytoplasmic polyadenylation elements (CPE). We found that miR-580, CPEB1, and CPEB2 act as negative regulators of TWIST1 expression in a sequence-specific and additive/cooperative manner.
