miRNAs as predictive biomarkers of obesity and weight lossepigenetic regulation and target gene identification

Resumen

Epigenetics refers to molecular factors and processes around the DNA that regulate genome activity without nucleotide sequence modification. One of these epigenetic factors are miRNAs, a class of small (17-25 nts in length) single stranded non coding RNAs that post transcriptionally regulate gene expression in animals, plants and unicellular eukaryotes. Furthermore, miRNAs coding regions may also be regulated by other epigenetic mechanisms, such as DNA methylation. Therefore, a complex interactive epigenetic regulation may occur onto DNA, ultimately controlling gene transcription and translation. Consequently, it has been demonstrated that epigenetics can be involved in the development of obesity, inflammation and metabolic disturbances (type 2 diabetes, hypercholesterolemia, hypertension, or cardiovascular disease). Furthermore, in the era of “personalized nutrition”, miRNAs have emerged as promising tools for the prediction, screening, diagnosis and prognosis of obesity and related comorbidities. In this sense, the present research work has applied different –omics approaches such as methylation/expression microarrays and deep sequencing technologies in order to address the following objectives: 1) to demonstrate the role of miRNA epigenetic regulation in the early stages of childhood obesity; 2) to identify miRNA-type biomarkers for weight loss prediction; and 3) to verify the regulation of selected miRNAs over their predicted target genes. In the first chapter we performed a methylation microarray in an infant population and identified that DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI SDS). Moreover, these methylation levels were able to explain up to 40% of the variation of BMI SDS in childhood obesity. In chapter 2, after combining methylation and expression microarrays data, we observed that miR-612 and miR-1976 were hypomethylated and overexpressed in high responders to a weight loss intervention. Additionally, after construction of specific expression vectors containing the particular 3’-UTR of each gene, we demonstrated that miR-612 and miR-1976 targeted TP53 and CD40 genes respectively. Furthermore, miR-1976 was able to down-regulate CD40 expression levels when transfected into mature human adipocytes. Lastly, in chapter 3, we noticed that miR-548q and miR-1185-1 also differed in their expression levels between high and low responders. Overexpression miRNA mimic experiments and luciferase reporter vectors showed that both miRNAs were implicated in GSK3B mRNA expression regulation, by a direct binding of the miRNA over the 3’ UTR of GSK3B in the case of miR-1185-1, and by an indirect regulation in the case of miR-548q. Overall, the results exhibited in this thesis add new insights into the importance of miRNAs in the management of diseases, especially of obesity and inflammation related illnesses; presenting different miRNAs involved in obesity development as prognostic biomarkers with potential utility in the personalization of weight loss treatments.