RESUMEN
miRNAs regulate post-transcriptional gene expression in metazoans, and thus are involved in many fundamental cellular biological processes. Extracellular miRNAs are also found in most human biofluids including plasma. These circulating miRNAs constitute a long distance inter cellular communication system and are potentially useful biomarkers. High throughput technologies like microarrays are able to scan a complete miRNome providing useful detection scores that are underexplored. We proposed to answer how many and which miRNAs are detectable in plasma or extracellular vesicles as these questions have not yet been answered. We set out to address this knowledge gap by analyzing the mirRNome in plasma and corresponding extracellular vesicles (EVs) from 12 children affected by retinoblastoma (Rb) a childhood intraocular malignant tumor, as well as from 12 healthy similarly aged controls. We calculated an average of 537 detectable miRNAs in plasma and 625 in EVs. The most miRNA enriched compartment were EVs from Rb cases with an average of 656 detectable elements. Using hierarchical clustering with the detection scores, we generated broad detection mirnome maps and identified a plasma signature of 19 miRNAs present in all Rb cases that is able to discriminate cases from controls. An additional 9 miRNAs were detected in all the samples; within this group, miRNA-5787 and miRNA-6732-5p were highly abundant and displayed very low variance across all the samples, suggesting both are good candidates to serve as plasma references or normalizers. Further exploration considering participant's sex, allowed discovering 5 miRNAs which corresponded only to females and 4 miRNAs corresponding only to males. Target and pathway analysis of these miRNAs revealed hormonal function including estrogen, thyroid signaling pathways and testosterone biosynthesis. This approach allows a comprehensive unbiased survey of a circulating miRNome landscape, creating the possibility to define normality in mirnomic profiles, and to locate where in these miRNome profiles promising and potentially useful circulating miRNA signatures can be found.
Asunto(s)
Vesículas Extracelulares/metabolismo , MicroARNs/sangre , Neoplasias de la Retina/patología , Retinoblastoma/patología , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Preescolar , MicroARN Circulante/sangre , Análisis por Conglomerados , Análisis Discriminante , Femenino , Humanos , Lactante , Masculino , MicroARNs/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Retina/genética , Retinoblastoma/genéticaRESUMEN
BACKGROUND: miRNAs exert their effect through a negative regulatory mechanism silencing expression upon hybridizing to their target mRNA, and have a prominent position in the control of many cellular processes including carcinogenesis. Previous miRNA studies on retinoblastoma (Rb) have been limited to specific miRNAs reported in other tumors or to medium density arrays. Here we report expression analysis of the whole miRNome on 12 retinoblastoma tumor samples using a high throughput microarray platform including 2578 mature miRNAs. METHODS: Twelve retinoblastoma tumor samples were analyzed using an Affymetrix platform including 2578 mature miRNAs. We applied RMA analysis to normalize raw data, obtained categorical data from detection call values, and also used signal intensity derived expression data. We used Diana-Tools-microT-CDS to find miRNA targets and ChromDraw to map miRNAs in chromosomes. RESULTS: We discovered a core-cluster of 30 miRNAs that were highly expressed in all the cases and a cluster of 993 miRNAs that were uniformly absent in all cases. Another 1022 miRNA were variably present in the samples reflecting heterogeneity between tumors. We explored mRNA targets, pathways and biological processes affected by some of these miRNAs. We propose that the core-cluster of 30 miRs represent miRNA machinery common to all Rb, and affecting most pathways considered hallmarks of cancer. In this core, we identified miR-3613 as a potential and critical down regulatory hub, because it is highly expressed in all the samples and its potential mRNA targets include at least 36 tumor suppressor genes, including RB1. In the variably expressed miRNA, 36 were differentially expressed between males and females. Some of the potential pathways targeted by these 36 miRNAs were associated with hormonal production. CONCLUSION: These findings indicate that Rb tumor samples share a common miRNA expression profile regardless of tumor heterogeneity, and shed light on potential novel therapeutic targets such as mir-3613 This is the first work to delineate the miRNA landscape in retinoblastoma tumor samples using an unbiased approach.
Asunto(s)
MicroARNs/genética , Retinoblastoma/genética , Transcriptoma , Adolescente , Adulto , Niño , Análisis por Conglomerados , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Retinoblastoma/patología , Factores Sexuales , Adulto JovenRESUMEN
El aumento en la sobrevida de los recién nacidos prematuros, las características del cuidado neonatal y la escasez de programas para la prevención, detección y tratamiento de la retinopatía del prematuro provocan que esta enfermedad sea la principal causa de ceguera infantil prevenible en México. El advenimiento de agentes antiangiogénicos de uso oncológico, y su uso -no autorizado, aunque con buenos resultados- en el tratamiento de enfermedades vaso proliferativas en la retina del paciente adulto, así como la presencia de reportes anecdóticos en la literatura y series de casos con serias fallas metodológicas han sugerido su utilización en el tratamiento de la retinopatía del prematuro. Desafortunadamente, estos agentes, utilizados indiscriminadamente, presentan absorción sistémica y causan efectos secundarios en el organismo del paciente prematuro. Además, no existen estudios de seguimiento a largo plazo que garanticen la seguridad de su uso en esta población. El presente artículo describe la situación en nuestro país y advierte sobre los riesgos de estos medicamentos en la población de pacientes prematuros.
The increase in survival rates among preterm infants, characteristics of neonatal care for such infants and a lack of suitable programs for preventing, detecting and treating retinopathy of prematurity (ROP) are factors that have made this disease the main cause of preventable blindness among children in Mexico. The advent of antiangiogenic agents in cancer treatment and their off-label use with favorable results in the treatment of proliferative vessel disease of the retina among adult patients, as well as anecdotal reports in the literature and a series of cases showing serious methodological flaws, have prompted their use in the treatment of retinopathy of prematurity. Unfortunately, these agents used indiscriminately in our country have a systemic absorption and secondary effects on the preterm patient's body. There are no long-term monitoring studies that guarantee their safe use in this segment of the population. This article describes the situation in our country and warns of the risks posed by the use of this type of drug on the preterm infant population.