EPIGENETIC MODIFICATIONS ABOLISH THE EXPRESSION OF THE LONG PENTRAXIN PTX3 IN HUMAN TUMORS.

PTX3 is a fluid-phase pattern recognition receptor that participates in innate immunity and inflammation by modulating complement activation, leukocyte recruitment, extracellular matrix deposition and angiogenesis. PTX3 is a biomarker of inflammatory conditions in different pathologies in humans, including acute myocardial infarction to autoimmune diseases, infections and cancer associated inflammation. Moreover, in vivo studies indicate that PTX3 is involved in cancer development, possibly by regulating inflammation. Several tumors lack PTX3 expression, such as human esophageal squamous cell carcinoma, where PTX3 is silenced through pro¬moter hypermethylation. Taken together, these data suggest that PTX3 is potentially involved in cancer development. The aim of this study was to identify the regulatory elements involved in the modulation of PTX3 expression and gain insight into their mechanisms of action in basal condition, inflammatory responses and cancer. Using in silico analysis of PTX3 gene we identified two putative PTX3 enhancers located 230 kbp upstream of the promoter and in the second exon of the gene, overlapping a CpG island, respectively. We performed ChIP assay for histone modifications and epigenetic complexes for the analysis of these genetic elements in human cell lines, before and after treatment with TNFα, that induces PTX3 expression. The results show that these regions are enhancers, but in basal condition are enriched with repressive markers H3K27me3 and polycomb group subunities (SUZ12 and EZH2). After TNFα treatment, the two enhancers became active gaining H3K27ac and the RNA Polymerase II and the first enhancer also acquired the binding for NF-kB. We also analysed the effect of the microRNAs, which were predicted to directly target PTX3, using bioinformatics analysis. miR-9, miR-29 family and miR-181 family were shown to directly target the PTX3 3’-UTR and to significantly reduce both PTX3 mRNA expression and protein production. Luciferase assay with PTX3 promoter and with a reporter vector for NF-kb showed that these miRNAs are also involved in signalling pathways controlling PTX3 transcription. Moreover, RNA Immuno Precipitation assay demonstrated that miR-9, miR-29 family and miR-181 family members and PTX3 mRNA were enriched in the RISC complex of macrophages after 6h of stimulation with lipopolysaccharide. Moreover, inflammatory miRNAs, such as miR-146a, miR-155 and miR-132 regulate PTX3 messenger and protein expression, as well, targeting the network upstream of PTX3 expression. This suggests that PTX3 gene is strictly regulated by several miRNAs acting at different points during inflammatory response. Bioinformatics analysis showed that PTX3 is not express in several cancer, including colorectal cancer (CRC). ELISA and gene expression analysis in CRC cells confirmed bioinformatics data and the treatment with the demethylating agent 5'aza-dC restored PTX3 expression and production. ChIP assay for histone modifications and transcription factors in CRC cells showed that PTX3 enhancers were inactive and they became active after treatment with 5’aza-dC and in response to inflammatory stimuli, thus acquiring the RNA polymerase II and NF-kB. Finally, by Methylated-CpG Island Recovery Assay we analysed the methylation of both promoter and enhancers of PTX3 in different CRC cells and in 40 CRC patients of different tumor stages. Data demonstrated that PTX3 regulatory regions were significantly methylated in all the cell lines and in all tumour stages, compared to their healthy counterparts. Moreover, the methylation started from the adenoma. Taken together, these data show that PTX3 methylation is involved in the inhibition of the PTX3 expression in CRC and suggest that PTX3 may have a protective role in the pathogenesis of this tumour.