RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Proteína HMGA1a/metabolismo , MicroARNs/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Proteína HMGA1a/química , Proteína HMGA1a/genética , Proteína HMGA1b/química , Proteína HMGA1b/genética , Proteína HMGA1b/metabolismo , Humanos , MicroARNs/química , MicroARNs/genética , Datos de Secuencia Molecular , Unión Proteica , Alineación de SecuenciaRESUMEN
High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64 approximately Lys-70 > Lys-66 > Lys-14 approximately Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70 approximately Lys-73 > Lys-64 approximately Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.
Asunto(s)
Proteína HMGA1a/metabolismo , Proteína HMGA1b/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Proteínas/metabolismo , Acetilación , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Proteína HMGA1a/química , Proteína HMGA1b/química , Histona Acetiltransferasas/química , Humanos , Masculino , Proteínas de Neoplasias/química , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The high mobility group A (HMGA) proteins are thought to work as ancillary transcription factors and to regulate the expression of a growing number of genes through direct binding to DNA or via protein-protein interactions. Both HMGA1 and HMGA2 are important regulators of basic biological processes, including cell growth, differentiation and transformation. Their qualitatively or quantitatively altered expression has been described in a number of human tumors. We studied and review here their expression in neuroblastic tumors. HMGA2 is expressed only in a subset of ex vivo neuroblastoma (NB) tumors and in the embryonic adrenal gland, but it is undetectable in the adult adrenal gland, suggesting that its anomalous expression might be associated with NB tumorigenesis and/or tumor progression. In vitro, its expression is easily detectable in retinoic acid (RA)-resistant cell lines. The exogenous expression of HMGA2 is sufficient to convert RA-sensitive SY5Y NB cells into RA-resistant cells, thus suggesting that HMGA2 might be a relevant player in determining NB cell responses to endogenous or therapeutically important growth inhibitory substances. In contrast, HMGA1 expression is readily detectable in all NB cell lines and tumors, but its expression is consistently higher in less differentiated NBs compared with ganglioneuromas and ganglioneuroblastomas. Interestingly, RA increases HMGA1 expression in RA-resistant NB cells but inhibits it in cells undergoing RA-induced growth inhibition and neuronal differentiation. Our studies indicate that HMGA molecules might be biologically and pathologically relevant factors in neuroblastic tumor development and progression.
Asunto(s)
ADN/química , Proteína HMGA1a/química , Proteína HMGA1b/química , Proteína HMGA2/química , Neuroblastoma/inmunología , Secuencia de Aminoácidos , Northern Blotting , Diferenciación Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Eliminación de Gen , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias/patología , Neuroblastoma/metabolismo , Fenotipo , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Transcripción Genética , Tretinoina/química , Tretinoina/metabolismoRESUMEN
High-mobility group (HMG) A1a and A1b proteins are among a family of HMGA proteins that bind to the minor groove of AT-rich regions of DNA. Here we employed tandem mass spectrometry and determined without ambiguity the sites of phosphorylation and the nature of methylation of HMGA1 proteins that were isolated from the PC-3 human prostate cancer cells. We showed by LC-MS/MS that Ser101 and Ser102 were completely phosphorylated in HMGA1a protein, whereas only a portion of the protein was phosphorylated at Ser98. We also found that the HMGA1b protein was phosphorylated at the corresponding sites, that is, Ser90, Ser91 and Ser87. In addition, Arg25, which is within the first DNA-binding AT-hook domain of HMGA1a, was both mono- and dimethylated. Moreover, both symmetric and asymmetric dimethylations were observed. The closely related HMGA1b protein, however, was not methylated. The unambiguous identification of the sites of phosphorylation and the nature of methylation facilitates the future examination of the biological implications of the HMGA1 proteins.