SINGLE NUCLEOTIDE POLYMORPHISMS AND MICRORNAS AFFECTING PTX3 PRODUCTION

Pentraxins are fluid phase pattern recognition receptors phylogenetically conserved from arachnids to mammals. Based on the primary structure, pentraxins are divided in short pentraxins, like the C-reactive protein and the serum amyloid P, and long pentraxins, like PTX3. PTX3 is expressed by several cell types, including mononuclear phagocytes, myeloid dendritic cells, fibroblasts, smooth muscle cells and endothelial cells in response to pro-inflammatory cytokines and microbial components. This molecule is involved in innate immunity, tissue remodelling, female fertility and in tuning the inflammatory response. In particular, PTX3 plays a protective role in cardiovascular diseases (CVD), such as acute myocardial infarction (AMI) and atherosclerosis, in preclinical studies. Moreover, PTX3 has emerged as novel diagnostic and prognostic biomarker in CVD, reflecting the inflammatory involvement of the vascular bed. In AMI patients, PTX3 levels peak 6-8 hours after the onset of symptoms and in this context it balances pro-inflammatory and antiinflammatory stimuli. Since single nucleotide polymorphisms (SNPs) and microRNAs represent two ways in which gene expression can be modified/regulated, we addressed their role in the modulation of PTX3 expression. Given that PTX3 gene SNPs have been reported to be associated to different clinical conditions, in particular in innate resistance to infections, we assessed the role of PTX3 SNPs in affecting PTX3 plasma levels and CVD susceptibility. Despite the characterized role of PTX3 in inflammation, a regulation of PTX3 by microRNAs, which are fundamental fine-tuners of this process, has not yet been described. Therefore, we investigated the role of microRNAs in regulating PTX3 expression. To our first aim, we performed a candidate-gene association study on Caucasian subjects, in about 1500 healthy individuals and 1700 AMI patients. PTX3 plasma levels were measured by ELISA in healthy subjects as well as in AMI patients from GISSI-Prevenzione trial. Blood was collected from AMI patients at least 5 days after the last event. A significant difference in PTX3 levels was detected between AMI patients and controls, suggesting the persistence of high PTX3 plasma levels in AMI patients up to three months after the last event. Moreover, in AMI patients, PTX3 plasma levels significantly correlated with mortality, but not with cardiovascular death or reinfarction, confirming the prognostic value of this parameter previously described as an independent predictor of 3-months mortality in this pathological condition. Moreover, we report that the 3 PTX3 SNPs analysed (the intronic rs2305619 and rs1840680 and the exonic rs3816527), alone or combined in haplotypes, are associated with different PTX3 plasma levels. However, we did not find a correlation between the 3 SNPs analysed and the clinical condition of the subject. About our second aim, data reported in this thesis reveal the existence of a complex network of microRNAs able to down-regulate the basal as well as the TNFα- and IL-1β-induced PTX3 production. The effect of microRNA over-expression was evaluated through the transfection of synthetic pre-miR in the human 8387 fibrosarcoma cell line, able to produce constitutively PTX3. The direct interaction miRNA:mRNA was evaluated through a luciferase reporter assay. Our results reveal that specific microRNAs, like miR-9 and miR-29, directly target and regulate PTX3 mRNA. Other microRNAs, including miR-29, impair PTX3 expression acting on molecules of the signalling pathway that leads to PTX3 transcription. Among these miRNAs there are also miR-146a and miR-155, two of the major microRNAs involved in controlling the inflammatory response. Another microRNA, miR-181c, impairs PTX3 production by targeting key molecules involved in PTX3 induction and by directly acting on the messenger of ERp18, a molecule involved in PTX3 folding. In conclusion, our results underline the complexity of PTX3 regulation, revealing that both PTX3 SNPs and microRNAs are fundamental players of this process.