Peptide Nucleic Acid (PNAs) are nucleic acid analogs in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone, usually formed by N-(2-amino-ethyl)-glycine units. PNAs show high binding affinity to RNA/DNA and are resistant to proteases and nucleases; moreover, PNAs are stable in cell culture conditions and in biological fluids. Accordingly, PNAs have been proposed as excellent candidates for in vitro and in vivo modulation of gene expression. The major limit of PNA-technology is low uptake by target cells; in order to solve this drawback, we linked PNAs to a polyarginine (R8) tails. MicroRNAs (miRs) are a family of small (19-25 nucleotides) noncoding RNAs regulating gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and the target sequences. In general, low expression of a given miRNA is linked with accumulation of targets mRNAs; conversely, high expression of miRNAs is expected to be the cause of a low expression of the target mRNAs. In this study we describe the activity of a PNA targeting miR-221, highly expressed in human breast cancer. A R8-PNA conjugate targeted against miR-221 (R8-PNA-a221) showed both very high affinity for RNA and efficient uptake within target cells without the need of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only R8-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by the PNA resulted in (a) lowering of the hybridization levels of miR-221 measured by RT-qPCR, (b) up-regulation of p27Kip1 mRNA and protein, measured by RT-qPCR and Western Blotting. Therefore, efficient delivery of anti-miR-221 PNAs through a suitable peptide carrier leads to inhibition of miR-221 activity, altering the expression of miR-221 regulated functions in breast cancer cells.
Peptide nucleic acids targeting miR-221 in breast cancer cell lines: uptake and modulation of miR-221 and p27kip1 biological function
BROGNARA, Eleonora;FABBRI, Enrica;BIANCHI, Nicoletta;FINOTTI, Alessia;BREVEGLIERI, Giulia;BORGATTI, Monica;GAMBARI, Roberto
2013
Abstract
Peptide Nucleic Acid (PNAs) are nucleic acid analogs in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone, usually formed by N-(2-amino-ethyl)-glycine units. PNAs show high binding affinity to RNA/DNA and are resistant to proteases and nucleases; moreover, PNAs are stable in cell culture conditions and in biological fluids. Accordingly, PNAs have been proposed as excellent candidates for in vitro and in vivo modulation of gene expression. The major limit of PNA-technology is low uptake by target cells; in order to solve this drawback, we linked PNAs to a polyarginine (R8) tails. MicroRNAs (miRs) are a family of small (19-25 nucleotides) noncoding RNAs regulating gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and the target sequences. In general, low expression of a given miRNA is linked with accumulation of targets mRNAs; conversely, high expression of miRNAs is expected to be the cause of a low expression of the target mRNAs. In this study we describe the activity of a PNA targeting miR-221, highly expressed in human breast cancer. A R8-PNA conjugate targeted against miR-221 (R8-PNA-a221) showed both very high affinity for RNA and efficient uptake within target cells without the need of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only R8-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by the PNA resulted in (a) lowering of the hybridization levels of miR-221 measured by RT-qPCR, (b) up-regulation of p27Kip1 mRNA and protein, measured by RT-qPCR and Western Blotting. Therefore, efficient delivery of anti-miR-221 PNAs through a suitable peptide carrier leads to inhibition of miR-221 activity, altering the expression of miR-221 regulated functions in breast cancer cells.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
