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Effects of pre-miR-93 and antagomiR-93 on morphology and apoptosis. A. Morphology of U251 glioma cells treated for 24 h with control, pre-miR-93 and antagomiR-93 molecules (200 nM). B,C. Effects of the different treatments on apoptosis on U251 and T98G glioma cell lines, as indicated. Apoptosis was analyzed by the Annexin-V release test [60] (B) or by caspase-3/7 production [61] (C). In panel B, the effects of a positive antagomiR-221 is also shown (see Brognara et al. (2014) [62]. Data represent the average S.D. of three independent experiments. ** = p

The study of new molecules able to selectively and stably interact with DNA and RNA is a field of great interest in consideration all the possible applications in medicine. Our study is related to the biomedical applications of the final product of PNA synthesis (the PNA itself) and the intermediate molecules obtained during the synthetic activity. In all the chemical synthesis approach of any pharmaceutical laboratory several molecules are produced, which are usually not considered for biological assays and technology transfer. We screened a set of C(5) uracil derivatives monomers, that were employed during the PNA synthesis, for activity on differentiated functions in K562 cells. We found that the highest antiproliferative effect and erythroid induction ability was exhibited by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine (compound 2) did not show any effect, suggesting the importance of the linear alkil chain in N(1) position. Compound 9, furthermore, exhibits induce erythroid terminal differentiation without activation of apoptotic pathway. The interest in the context on anti-tumor differentiation therapy is related to the fact that, when compared to known erythroid differentiation antitumor inducers (such as for instance cytosine arabinoside, mithramycin, resveratrol), the lead compound we were able to identify is the most active agent. Therefore these molecules in our opinion deserves further research activity in order to define its possible application, for instance in the control of proliferation/differentiation of CML primary cells resistance to the commonly employed Imatinib (Gleevec®) therapy. As far as the final product of the synthetic strategy (a PNA recognizing miR- 221 and able to be internalized in target tumor cells thanking to a linked Arg-8 peptide), the results here presented allows to conclude that (a) it is internalized at high efficiency into target tumor cells; (b) inhibits the miR-221 hybridization availability and (c) has important effects on biological functions regulated by miR-221 (i.e. expression of the p27Kip1 mRNA/protein). These results are in our opinion of interest, considering on one hand the role of miR-221 in cancer and, on the other hand, the role of p27Kip1. As far as miR-221, it was found to be up-regulated in several tumors; in breast cancer miR-221 is up-regulated in breast cancer cell line (such as MDA-MB-231) and in metastatic tumors. In the contest of breast cancer, it was identified p27kip1 mRNA as possible target of miR-221. The cyclin-kinase inhibitor p27kip1 is a tumorsuppressor protein, involved in the control of cell cycle during the G0/G1 check-point transition: the loss or decrease of p27kip1, together with others, is one of contributory causes of the “proliferating state” of invasive cancer, which remains in this growth phase without arrive to differentiation. It is of great interest since it was found to be down-regulated in several type of tumors. The studies presented in this PhD Thesis teach that even intermediate synthetic molecules deserve attention in respect to possible biological effects on cells relevant to human pathologies. Moreover, we have identify two reagents of possible interest for the development of anti-cancer protocols: N(1)-octyl-thymine for the treatment of CML cells and Rpep-PNA-a221 for possible use in the treatment of breast cancer cells.

International audience; In several types of thalassemia (including β039-thalassemia) stop codon mutations lead to premature translation termination and to mRNA destabilization through non-sense mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal read-through. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In the present study we have employed a lentiviral construct containing the β039-thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by FACS analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and HPLC analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal read-through should be considered a strategy for developing experimental strategies for treatment of β0-thalassemia caused by stop codon mutations.