Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5'-5' inversion of polarity site, namely d(3'GGT5'-5'TGGTGTGGTTGG3'), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3'GGT5'-5'TGGTGTGGTTGG3') is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4 degrees C higher than the corresponding unmodified TBA. d(3'GGT5'-5'TGGTGTGGTTGG3') continues to display an anticoagulant activity, even if decreased with respect to the TBA.

NMR, molecular dynamics and mechanics calculations, and CD spectroscopy were used to characterise three tetramolecular quadruplex complexes: [d(TG^BrGGT)]₄, [d(TGG^BrGT)]₄ and [d(TGGG^BrT)]₄, where G^Br indicates an 8-bromoguanine residue. All three quadruplexes are characterised by a 4-fold symmetry with all strands parallel to each other and, differently to what has been observed for other parallel quadruplex structures, with a tetrad (formed by 8-Br-dGs) in a syn conformation. The whole of the data demonstrates that the replacement in turn of different dG residues with 8-Br-dG in the sequence 5′-TGGGT-3′ affects the resulting structures in different ways, leading to different CD profiles and thermal stabilities. Particularly, [d(TG^BrGGT)]₄ and [d(TGG^BrGT)]₄ are more stable than the unmodified sequence, whereas [d(TGGG^BrT)]₄ is much less stable than the natural counterpart. The conformational features found in the three quadruplexes might, in principle, amplify the range of applicability of synthetic oligonucleotides as aptamers or catalysts, by providing novel structural motifs with different molecular recognition capabilities from those of native DNA sequences.