Cuantificación de microRNAS en neonatos con normopeso,bajo peso y macrosomia usando tarjetas de tamiz neonatal

Tesis (Maestría en Ciencias en Biología Molecular)"El peso al nacimiento es un indicador temprano de enfermedades metabólicas y los microRNAs (miRNAs) son reguladores post-transcripcionales relacionados con diabetes y obesidad. En este trabajo estandarizamos un protocolo para extraer y cuantificar miRNAs a partir de muestras de sangre de tamiz neonatal almacenada en tarjetas de Guthrie y analizamos la expresión de los miRNAs hsa-miR-375, hsamiR-33b y hsa-miR-454-3p de neonatos con normopeso (n=20), bajo peso (n=20) y macrosomía (n=20). La expresión de hsa-miR-375 no tuvo diferencias significativas en los tres grupos (p=0.153), pero hsa-miR-454-3p se sobreexpresó en neonatos con peso bajo (p<0.001) y hsa-miR-33b se sobreexpresó en neonatos macrosómicos (p<0.001). Para explorar el posible papel de los miRNAs sobreexpresados sobre la programación fetal de enfermedades metabólicas, mediante análisis bioinformático encontramos que los posibles genes blanco se asocian a procesos como neurogénesis, homeostasis de glucosa, metabolismo de sustancias orgánicas y respuesta celular a estímulos nutricionales (p<0.01). Nuestros resultados constituyen la primera evidencia de alteraciones en la expresión de miRNAs en neonatos con peso alto y bajo al nacimiento y abren una promisoria área de investigación sobre los mecanismos moleculares de la programación fetal.""Birth weight is an early predictor for metabolic diseases and microRNAs (miRNAs) are post-transcriptional regulators related to diabetes and obesity. In this work we standardized a protocol for miRNAs extraction and quantification from neonatal screening blood samples stored in Guthrie cards and analyzed the expression of miRNAs hsa-miR-375, hsa-miR-33b and hsa-miR-454-3p in normal birth weight (n=20), low birth weight (n=20) and macrosomic (n=20) neonates. hsa-miR-375 expression did not vary significantly among groups (p=0.153), whereas hsa-miR-454-3p was over-expressed in low birth weight neonates (p<0.001) and hsa-miR-33b was over-expressed in macrosomic neonates (p<0.001). To explore the possible role of the over-expressed miRNAs on the fetal programming of metabolic diseases, through bioinformatic analysis we found their potential target genes are associated to processes such as neurogenesis, glucose homeostasis, organic substance metabolism and cellular response to nutrient stimulus (p<0.01). Our results constitute the first evidence of altered miRNAs expression in neonates with high and low birth weight and open up a promising research area on the molecular mechanisms of fetal programming.

Newborn expression of microRNAs as potential early biomarkers for metabolic syndrome

"El síndrome metabólico es un trastorno caracterizado por obesidad, hipertensión arterial, hipercolesterolemia, hiperlipidemia y alteraciones en el metabolismo de la glucosa, y constituye un factor de riesgo para el desarrollo de diabetes y enfermedades cardiovasculares. Las condiciones nutricionales, hormonales y metabólicas de la madre durante el embarazo y el posparto temprano pueden condicionar el desarrollo de enfermedades metabólicas en la vida adulta de los hijos. Este concepto, llamado “programación fetal”, sugiere la existencia de marcadores moleculares que permitirían estimar de forma temprana el riesgo de desarrollar síndrome metabólico e implementar medidas para prevenirlo. Este trabajo revisa el potencial de los microRNAs como biomarcadores para la detección temprana del síndrome metabólico dada su especificidad y estabilidad, su presencia en fluidos biológicos de obtención rutinaria en la clínica y su expresión temprana en diversas enfermedades.""Metabolic syndrome is a disorder characterised by obesity, hypertension, hypercholesterolemia, hyperlipidemia and alterations in glucose metabolism. It constitutes a risk factor for diabetes and cardiovascular disease development. Nutritional, hormonal and metabolic conditions of the mother during pregnancy and the early life of the offspring can determine metabolic disease development during the offspring's later life. This concept, called “fetal programming”, suggests the possibility of finding molecular markers to make early estimations for the risk of developing metabolic syndrome and of working on prevention strategies. This paper reviews the potential of microRNAs as early detection biomarkers for metabolic syndrome, given that they are specific and stable, are present in body fluids routinely obtained in clinics and have been found in early stages of several diseases.

Analysis of microRNA expression in newborns with differential birth weight using newborn screening cards

"Birth weight is an early predictor for metabolic diseases and microRNAs (miRNAs) are proposed as fetal programming participants. To evaluate the use of dried blood spots (DBS) on newborn screening cards (NSC) as a source of analyzable miRNAs, we optimized a commercial protocol to recover total miRNA from normal birth weight (NBW, n= 17–20), low birth weight (LBW, n = 17–20) and high birth weight (macrosomia, n = 17–20) newborns and analyzed the relative expression of selected miRNAs by stem-loop RT-qPCR. The possible role of miRNAs on the fetal programming of metabolic diseases was explored by bioinformatic tools. The optimized extraction of RNA resulted in a 1.2-fold enrichment of miRNAs respect to the commercial kit. miR-33b and miR-375 were overexpressed in macrosomia 9.8-fold (p < 0.001) and 1.7-fold, (p < 0.05), respectively and miR-454-3p was overexpressed in both LBW and macrosomia (19.7-fold, p < 0.001 and 10.8-fold, p < 0.001, respectively), as compared to NBW. Potential target genes for these miRNAs are associated to cyclic-guanosine monophosphate (cGMP)-dependent protein kinase (PKG), mitogen-activated protein kinase (MAPK), type 2 diabetes, transforming growth factor-β (TGF-β)and Forkhead box O protein (FoxO) pathways. In summary, we improved a protocol for analyzing miRNAs from NSC and provide the first evidence that birth weight modifies the expression of miRNAs associated to adult metabolic dysfunctions. Our work suggests archived NSC are an invaluable resource in the search for fetal programming biomarkers.