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Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221

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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 oligoarginine 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 p27Kip1 (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27Kip1 mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27Kip1 mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27Kip1 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.

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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

TIMP3:

Metalloproteinase inhibitor 3

ICAM-1:

Intercellular adhesion molecule 1

SPR:

Surface plasmon resonance

HBS:

Hepes buffered saline

Ab:

Antibody

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Acknowledgements

This work was supported by grants by AIRC (contract number IG13575) and by MIUR (PRIN09). We are grateful to Drs. Giuseppe Moretto, Alberto Beltramello, Antonio Iannucci, Giampietro Pinna, Mario Meglio, Antonio Nicolato, Bruno Bonetti, Maria Cristina Dechecchi, the Colleagues of the Dept. of Neurosciences and the Dept. of Pathology and Diagnostics of the Hospital of Verona for helpful discussions and to Dr. Amanda J. Neville for kindly revising the English text. Cinzia Cantù is a fellow of the Banco Popolare di Verona, Verona, Italy.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy

    Eleonora Brognara, Enrica Fabbri, Giulia Montagner, Nicoletta Bianchi, Alessia Finotti, Giulia Breveglieri, Monica Borgatti & Roberto Gambari

  2. Genetic Unit, IRCCS Centro San Giovanni di Dio, FBF, Brescia, Italy

    Elena Bazzoli

  3. Department of Pathology and Diagnostics, University Hospital of Verona, Verona, Italy

    Claudio Ghimenton, Albino Eccher, Valentino Bezzerri & Giulio Cabrini

  4. Department of Chemistry, University of Parma, Parma, Italy

    Alex Manicardi & Roberto Corradini

  5. Laboratory of Molecular Pathology, University-Hospital, Verona, Italy

    Cinzia Cantù, Valentino Bezzerri & Giulio Cabrini

  6. Department of Neurological, Neuropsychological, Morphological and Movement Sciences, University of Verona, Verona, Italy

    Elena Bazzoli

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  1. Eleonora Brognara
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Correspondence to Roberto Gambari.

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Brognara, E., Fabbri, E., Bazzoli, E. et al. Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221. J Neurooncol 118, 19–28 (2014). https://doi.org/10.1007/s11060-014-1405-6

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