LABORATORY INVESTIGATION Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221 Eleonora Brognara • Enrica Fabbri • Elena Bazzoli • Giulia Montagner • Claudio Ghimenton • Albino Eccher • Cinzia Cantu ` • Alex Manicardi • Nicoletta Bianchi • Alessia Finotti • Giulia Breveglieri • Monica Borgatti • Roberto Corradini • Valentino Bezzerri • Giulio Cabrini • Roberto Gambari Received: 20 August 2013 / Accepted: 17 February 2014 / Published online: 5 March 2014 Ó Springer Science+Business Media New York 2014 Abstract MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a olig- oarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR- 221. In addition, the effects of R8-PNA-a221 on p27 Kip1 (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27 Kip1 mRNA content occurs in U251 cells in the pre- sence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27 Kip1 mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27 Kip1 protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR- 221 might be proposed for experimental treatment of human gliomas. Keywords Peptide nucleic acids Á Glioma Á MicroRNAs Á MiR-221 Á P27 Kip1 Á MiRNA targeting Á Delivery Abbreviations PNA Peptide nucleic acid Fl Fluorescein RT-qPCR Reverse transcription quantitative polymerase-chain reaction SDS Sodium dodecylsulphate SDS-PAGE SDS-polyacrylamide gel electrophoresis TMZ Temozolomide PUMA p53-upregulated modulator of apoptosis PTEN Phosphatase and tensin homolog Electronic supplementary material The online version of this article (doi:10.1007/s11060-014-1405-6) contains supplementary material, which is available to authorized users. E. Brognara Á E. Fabbri Á G. Montagner Á N. Bianchi Á A. Finotti Á G. Breveglieri Á M. Borgatti Á R. Gambari (&) Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy e-mail: gam@unife.it E. Bazzoli Genetic Unit, IRCCS Centro San Giovanni di Dio, FBF, Brescia, Italy E. Bazzoli Department of Neurological, Neuropsychological, Morphological and Movement Sciences, University of Verona, Verona, Italy C. Ghimenton Á A. Eccher Á V. Bezzerri Á G. Cabrini Department of Pathology and Diagnostics, University Hospital of Verona, Verona, Italy C. Cantu ` Á V. Bezzerri Á G. Cabrini Laboratory of Molecular Pathology, University-Hospital, Verona, Italy A. Manicardi Á R. Corradini Department of Chemistry, University of Parma, Parma, Italy 123 J Neurooncol (2014) 118:19–28 DOI 10.1007/s11060-014-1405-6