Capobianco, Valentina (2015) Epigenetic and proteomic characterization in human morbid obesity. [Tesi di dottorato]

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Abstract

Obesity is an epidemic health problem worldwide associated with increased risk of cardiovascular disease, metabolic syndrome, and cancer. Its incidence increased in pregnant women in the last two decades as well as observed in the general population. Maternal obesity is related to offspring obesity, and there is an increased risk of adverse outcomes for both mother and child. Visceral adipose tissue (VAT) is an important risk factor for metabolic imbalance in human subjects, also during pregnancy. So, our aim was to study epigenetic regulation and proteomic signature of obesity in morbid obese women with and without pregnancy. The first aim of this study was to investigate the miRNA-expression profile and the proteomic signature in VAT from obese women to identify miRNA/protein target pairs associated with obesity. Notably, most miRNAs were down-expressed in obese tissues, whereas most of the proteins from the investigated spots were up-expressed. Bioinformatics integration of miRNA expression and proteomic data highlighted two potential miRNA/protein target pairs: miR-141/YWHAG (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide) and miR-520e/RAB11A (Ras-related protein RAB-11A); the functional interaction between these miRNAs and their target sequences on the corresponding mRNAs was confirmed by luciferase assays. Both RAB11A and YWHAG proteins are involved in glucose homeostasis; YWHAG is also involved in lipid metabolism. Hence, the identified miRNA/protein target pairs are potential players in the obese phenotype. The second aim of this study was to investigate the effects of maternal pre-pregnancy obesity in placental tissue and in human amniotic mesenchymal stem cells (hA-MSCs) from morbidly obese women to highlight differential expression patterns to correlate with the obese phenotype. The miRNA-expression profile was studied in amnion from obese and control women. Seven miRNAs were expressed only in amnion from obese women, whereas 13 miRNAs were up-expressed and 12 miRNAs down-expressed in amnion from obese women compared to controls. Target genes of these miRNAs and miRNA-regulated pathways were predicted by bioinformatics. MiRNAs significantly down-regulated the neurotrophin, cancer/ErbB, mammalian target of rapamycin, insulin, adipocytokine, actin cytoskeleton and mitogen-activated protein kinase signaling pathways. In conclusion, this study shows that the miRNA profile is altered in amnion during obesity and we hypothesize that this could affect pathways important for placental growth and function, thereby contributing to an increase in the newborn's risk of future metabolic diseases. In hA-MSCs from obese (Ob-) and non-obese (Co-) pregnant women were studied both the miRNA and protein expression profiles to highlight differential expression patterns to correlate with the obese phenotype. Among the tested miRNAs 11 were up-expressed and 14 were down-expressed in Ob- compared to Co-hA-MSCs. Interestingly, 7 miRNAs were obesity-specific, being expressed only in Ob-hA-MSCs. Bioinformatics showed that differently expressed miRNAs significantly regulated genes belonging to several metabolic pathways, i.e. MAPK signalling, actin cytoskeleton, focal adhesion, axon guidance, insulin signaling, etc. Proteomic signature showed 40 differently expressed protein spots, 62% were increased and 38% were decreased in Ob- compared to Co-hA-MSCs. Globally, a total of 41 proteins were identified in these spots. They were involved into 5 pathways: Focal adhesion, Processing in endoplasmic reticulum, Metabolic pathways, Regulation of actin cytoskeleton, MAPK signaling. Further investigations are needed to validate proteomic data and to identify miRNA/protein target pairs in hA-MSCs. In conclusion, these data highlight in Ob-hA-MSCs altered pathways that were relevant for both metabolic function and structural integrity. Interestingly, these pathways were previously found to be altered in whole placenta or in adipose tissue from obese women, so supporting that cellular dysfunctions are present in utero during obesity and likely contribute to increase the newborns' risk for metabolic diseases in adult life.

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