RESUMO
BACKGROUND INFORMATION: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16. RESULTS: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. CONCLUSIONS: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Proteína HMGA1a/metabolismo , MicroRNAs/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Proteína HMGA1a/química , Proteína HMGA1a/genética , Proteína HMGA1b/química , Proteína HMGA1b/genética , Proteína HMGA1b/metabolismo , Humanos , MicroRNAs/química , MicroRNAs/genética , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de SequênciaAssuntos
Proteínas de Ligação a DNA/genética , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Adolescente , Adulto , Southern Blotting , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Cariotipagem , Proteína de Leucina Linfoide-Mieloide , Translocação GenéticaRESUMO
Signaling of TGFbeta family members such as activin is tightly regulated by soluble binding proteins. Follistatin binds to activin A with high affinity, and prevents activin binding to its own receptors, thereby blocking its signaling. We previously identified FLRG gene from a B-cell leukemia carrying a t(11;19)(q13;p13) translocation. We and others have already shown that FLRG, which is highly homologous to follistatin, may be involved in the regulation of the activin function through its binding to activin. In this study, we found that, like follistatin, FLRG protein inhibited activin A signaling as demonstrated by the use of a transcriptional reporter assay, and blocked the activin A-induced growth inhibition of HepG2 cells. We have recently shown that the TGFbeta-induced expression of FLRG occurs at a transcriptional level through the action of Smad proteins. Here we show that activin A increases FLRG and follistatin at both the mRNA and protein levels. We found that Smad proteins are involved in the activin A-induced transcription activation of FLRG and follistatin. Finally we demonstrate that FLRG protein regulates its own activin-induced expression. In conclusion, activin A induces FLRG and follistatin expression. This observation, in conjunction with the antagonistic effect of FLRG and follistatin on activin signaling, indicates that these two proteins participate in a negative feedback loop which regulates the activin function.
