RESUMEN
MicroRNAs (miRNAs), small 22-25 nucleotide non-coding RNAs, play important roles in cellular and tumor biology. However, characterizing miRNA function remains challenging due to an abundance of predicted targets and an experimental bottleneck in identifying biologically relevant direct targets. Here, we developed a novel technique (miFAST) to identify direct miRNA target genes. Using miFAST, we confirmed several previously reported miR-340 target genes and identified five additional novel direct miR-340 targets in melanoma cells. This methodology can also be efficiently applied for the global characterization of miRNA targets. Utilizing miFAST to characterize direct miRNA targetomes will further our understanding of miRNA biology and function.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Biología Molecular/métodos , Línea Celular Tumoral , Perfilación de la Expresión Génica/instrumentación , Humanos , Biología Molecular/instrumentación , Reproducibilidad de los ResultadosRESUMEN
Alternative cleavage and polyadenylation generates multiple transcript variants producing mRNA isoforms with different length 3'-UTRs. Alternative cleavage and polyadenylation enables differential post-transcriptional regulation via the availability of different cis-acting elements in 3'-UTRs. Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and melanogenesis. This central transcription factor is also implicated in melanoma development. Here, we show that melanoma cells favor the expression of MITF mRNA with a shorter 3'-UTR. We also establish that this isoform is regulated by a micro RNA (miRNA/miR), miR-340. miR-340 interacts with two of its target sites on the MITF 3'-UTR, causing mRNA degradation as well as decreased expression and activity of MITF. Conversely, the RNA-binding protein, coding region determinant-binding protein, was shown to be highly expressed in melanoma, directly binds to the 3'-UTR of MITF mRNA, and prevents the binding of miR-340 to its target sites, resulting in the stabilization of MITF transcripts, elevated expression, and transcriptional activity of MITF. This regulatory interplay between RNA-binding protein and miRNA highlights an important mechanism for the regulation of MITF in melanocytes and malignant melanomas.
Asunto(s)
Regiones no Traducidas 3' , Regulación Neoplásica de la Expresión Génica , Melanocitos/metabolismo , MicroARNs/genética , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas de Unión al ARN/genética , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células HEK293 , Humanos , Melanocitos/patología , MicroARNs/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Transducción de SeñalRESUMEN
microRNA (miRNA)-dependent regulation of gene expression is increasingly linked to development and progression of melanoma. In this study we evaluated the functions of miR-340 in human melanoma cells. Here, we show that miR-340 inhibits the tumorigenic phenotype of melanoma cells. We also found that miR-340 regulates RAS-RAF-Mitogen Activated Protein Kinase (MAPK) signaling by modulating the expression of multiple components of this pathway. Given the importance of MAPK signaling in melanoma, these results provide further insight into the pathogenesis of melanoma.