IL RUOLO DEI miRNA NEL PDAC: analisi funzionale di miR-216a-5p e miR-125a-5p nelle Cellule Staminali Tumorali pancreatiche.

The present thesis derives from 3-year phD project focused on biomarker discovery for Pancreatic Ductal Adenocarcinoma diagnosis and prognosis. Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to late-stage diagnoses resulting from nonspecific early symptoms and the absence of early diagnostic biomarkers. MicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, with their abnormal expression observed in various diseases, including cancer. The experimental workflow has been divided into two sections. The first, exploratory, merges in silico, ex vivo and in vitro experiments to identify PDAC-associated miRNAs. The second part is a functional study aimed at characterizing the effects of miR-216a-5p and miR-216b-5p in the acquisition or regression of Cancer Stem Cells (CSCs) traits. Initially, miRNA profiling datasets from GeoDataset underwent a meta-analysis, revealing a set of PDAC-associated miRNAs. The panel was then investigated in serum and solid biopsies of 37 PDAC patients, as well as in multiple PDAC cellular models. The focus was extended towards exploring the possible role of PDAC-associated microRNAs to the (CSC) phenotype, by inducing CSC-rich pancreatospheres from PDAC cell lines. Then, the role of miR-216a-5p and miR-125a-5p in the expression of CSC markers, pluripotency genes, and EMT genes, as well as in clonogenic activity and cellular vitality, was evaluated in adherent BxPC-3 and AsPC-1 cells and their CSC-like models following transient transfection. Meta-analysis showed differentially expressed miRNAs in blood samples and cancerous tissues of patients, partially aligning with our patient data. In vitro tasks confirmed the aberrant expression of PDAC-associated miRNAs. Globally, our results suggest that miR-216a-5p and miR-216b-5p could exert a dual role in PDAC while miR-4486, miR-361-3p, miR-125a-5p, miR-320d could promote PDAC initiation and progression. MiR-125a-5p demonstrated a promoting role for the CSC phenotype by enhancing the expression of CD44/CD24/CxCR4 markers and Nanog, Oct-4, and Sox2 genes. MiR-216a-5p exhibited a noteworthy behavior that partially validated the previous hypothesis regarding its possible dual function. This study contributed to an enhanced comprehension of the role of miRNAs in the development and progression of PDAC. Furthermore, it sheds new light on the miRNA landscape in PDAC and its intricate interplay with CSCs, providing insights for diagnostic and therapeutic improvements.

The present thesis derives from 3-year phD project focused on biomarker discovery for Pancreatic Ductal Adenocarcinoma diagnosis and prognosis. Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to late-stage diagnoses resulting from nonspecific early symptoms and the absence of early diagnostic biomarkers. MicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, with their abnormal expression observed in various diseases, including cancer. The experimental workflow has been divided into two sections. The first, a discovery study, merges in silico, ex vivo and in vitro experiments to identify PDAC-associated miRNAs. The second part is a functional study aimed at characterizing the effects of miR-216a-5p and miR-125a-5p in the acquisition or regression of Cancer Stem Cells (CSCs) traits. Initially, miRNA profiling datasets from GeoDataset underwent a meta-analysis, revealing a set of PDAC-associated miRNAs. The panel was then investigated in serum and solid biopsies of 37 PDAC patients, as well as in multiple PDAC cellular models. The focus was extended towards exploring the possible role of PDAC-associated microRNAs to the (CSC) phenotype, by inducing CSC-rich pancreatospheres from PDAC cell lines. Then, the role of miR-216a-5p and miR-125a-5p in the expression of CSC markers, pluripotency genes, and EMT genes, as well as in clonogenic activity and cellular vitality, was evaluated in adherent BxPC-3 and AsPC-1 cells and their CSC-like models following transient transfection. Meta-analysis showed differentially expressed miRNAs in blood samples and cancerous tissues of patients, partially aligning with our patient data. In vitro tasks confirmed the aberrant expression of PDAC-associated miRNAs. Globally, our results suggest that miR-216a-5p and miR-216b-5p could exert a dual role in PDAC while miR-4486, miR-361-3p, miR-125a-5p, miR-320d could promote PDAC initiation and progression. MiR-125a-5p demonstrated a promoting role for the CSC phenotype by enhancing the expression of CD44/CD24/CxCR4 markers, Oct-4, and Sox2 genes. MiR-216a-5p exhibited a noteworthy behavior that partially validated the previous hypothesis regarding its possible dual function. This study contributed to an enhanced comprehension of the role of miRNAs in the development and progression of PDAC. Furthermore, it sheds new light on the miRNA landscape in PDAC and its intricate interplay with CSCs, providing insights for diagnostic and therapeutic improvements