miRNAs are oligonucleotides acting as negative regulators of gene expression; the aim of our study was to verify their role in the regulation of genes involved in two specific pathological conditions: celiac disease and hypoxia. Celiac disease (CD) is an autoimmune disease of the intestine that culminates in villous atrophy. We performed a microarray analysis comparing the duodenum of adult CD patients to that of healthy subjects. We detected the down-regulation of 7 miRNAs; 4 of them were confirmed by qRT-PCR, and their level correlated with the gravity of the mucosal damage. In silico analyses identified the target genes of these miRNAs, proteins involved in the immune response. in particular miR-192-5p acts on NOD2 and CXCL2, miR-31-5p on FOXP3, whereas miR-338 and miR-197 target RUNX1 and IL18, respectively. mRNA and protein levels of all targets resulted increased in CD patients compared to controls, and a significant inverse correlation with the respective miRNA was observed. Clinical manifestations of CD are very heterogeneous and age-dependent, thus we hypothesized that the underlying molecular mechanisms could be influenced by age. We analyzed the same miRNAs and target genes in pediatric CD patients compared to controls and the results showed the same trend of expression observed in adult ones, except for NOD2 and CXCL2 that did not show any expression variation. On the contrary, another miR-192-5p target, MAD2L1, involved in the cell cycle and intestinal mucosa remodeling, resulted up-regulated only in CD pediatric patients whereas it did not show any variations in adults. These data support our hypothesis on the presence of age-dependent molecular mechanisms. Finally, we hypothesized that a panel of plasmatic miRNAs could improve the current diagnostic tools; we analyzed the same miRNAs in the plasma of CD subjects detecting a trend of expression similar to that observed in the biopsies, but in patients on a gluten free diet we did not observe a return to normal. The other analyzed pathological condition is hypoxia, characterized by a reduction of oxygen levels, and observed in various situations including anemia. We focused our attention on the mechanisms of iron homeostasis in these conditions, hypothesizing a role played by miRNAs. We analyzed the liver of mice exposed to hypoxia (12, 24 and 48 hours) and compared to mice on normoxia. We detected a down-regulation of the entire Bmp/Smad pathway, in particular we analyzed gene and protein expression of Bmp6, HJV, BmpR1a, BmpR2, Smad1/5/8, Id1 and Hamp, the main regulator of iron homeostasis. Hypothesizing a role of miRNAs, we performed a microarray analysis on the liver of mice exposed to hypoxia, identifying several differentially expressed miRNAs. We verified by qRT-PCR the expression of miR-22-3p, miR-101a-3p e miR-351 that resulted up-regulated according to the time of exposure and inversely correlated with their targets Bmp6, BmpR1a and Atoh8 (Hamp transcriptional regulator), respectively. In conclusion, we suggested the pivotal role hypoxia-dependent of miRNAs in the regulation of genes involve in iron metabolism and in particular of Bmp/Smad pathway.
|Data di pubblicazione:||4-mar-2016|
|Titolo:||Gene expression regulation microRNA-dependent in two different pathological conditions: celiac disease and hypoxia|
|Settore Scientifico Disciplinare:||BIO/13 - BIOLOGIA APPLICATA|
|Corso di dottorato:||TECNOLOGIE BIOMEDICHE - 20R|
|Citazione:||(2016). Gene expression regulation microRNA-dependent in two different pathological conditions: celiac disease and hypoxia. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).|
|Parole Chiave (Inglese):||microRNA|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|