Studio dei meccanismi molecolari alla base dell’azione dei microRNA in processi patologici

Abstract

My research project concerns the study of the molecular mechanisms involving microRNAs (miRNAs) in pathological processes such as tumors and cardiovascular diseases. miRNAs are small non-coding RNAs (sncRNAs) acting by modulating post-transcriptionally gene expression of specific mRNA target. Many studies have shown that the aberrant expression of these sncRNA is closely correlated with the onset of many diseases, including cancer and occlusive cardiovascular syndromes. Therefore, the study was conducted on three different experimental models such as activated platelets in vitro, tumor tissues of endometrial cancer (EC) and breast cancer (BC) cells expressing the estrogen receptor β (ERβ). Platelet activation is involved not only in physiological processes as haemostasis but also in the thrombogenesis and the pathogenesis of occlusive cardiovascular disorders including Acute Coronary Syndromes (ACS). However, the several events of resistance to antithrombotic therapies suggest the existence of mechanisms, at the basis of the activation process, still not known. The data that I obtained, following ex vivo activation of platelets from healthy donors, demonstrated the existence of miRNA pathway maturation in platelets and a significant reorganization of platelet proteome mediated by miRNAs upon activation. EC is one of the most frequent types of cancer affecting the female population and devopls, in most of cases, from an hyperplastic condition. I identified changes in the expression of miRNAs and other sncRNAs from paired normal, hyperplastic and cancerous endometrial tissues obtained by hysteroscopy. The results obtained allowed me to define a signature of sncRNAs deregulated in neoplastic transformation, characterized by 129 miRNAs, 10 piRNAs (PIWI-interacting RNAs) and 3 snoRNAs (small nucleolar RNAs). Moreover, in silico analysis of downstream targets revealed that these endometrial sncRNAs are involved in several cellular processes and canonical pathways, including TGF-β, ERK/MAPK and Wnt/β-catenin signaling pathways. In 70% of the cases BC is a hormone-responsive tumor since cells are able to respond to proliferative stimuli induced by estrogens thanks to estrogen receptors (ERs). Recent studies have shown that unliganded-ERβ is equally distributed between the nucleus and cytoplasm and that its presence determines a reduction of cell growth. In this study, by applying gene expression profiling and in vivo global mapping of ERβ binding site to genome, I have demonstrated that the receptor is able to regulate gene expression at transcriptional level also in the absence of ligand. The results obtained from smallRNA-seq experiments revealed that miRNAs expression profile is influenced by costitutive-ERβ activity in MCF-7 BC cells. Proteomic analysis of molecular partners of unliganded ERβ showed an association with Argonaute 2 (AGO2). RNA immunoprecipitation coupled to massive parallel sequencing (RIP-Seq) experiments revealed that ERβ-AGO2/miRNA complex is able to bind and regulate at post-transcriptional level specific mRNAs involved in several biological pathways in BC cells deprived of hormonal stimuli. [edited by author]