MicroRNAs (miRNAs) are an abundant class of naturally occurring, small non-coding RNA molecules that measure ~19-25 nucleotides in length able to regulate gene expression at the post-transcriptional level by (i) cleavage of target mRNAs to induce their degradation or alternatively by (ii) translational repression of protein-coding genes, depending on the degree of complementarity between miRNAs and the target sequences. In cancer, miRNAs play a double role: miRs exhibiting tumor-suppressor properties usually target mRNAs coding oncoproteins, while cancer-promoting miRs (onco-miRNAs) target mRNA coding for tumor-suppressor proteins. The oncomiR miR-221 is highly expressed in human breast cancer. MDA-MB-231 and MCF-7 cells were first characterized with respect to expression of miR-221 and p27Kip1. Our experiments (done by RT-qPCR) confirm that miR-221 is up-regulated in MDA-MB-231 cells in respect to MCF-7 cells and, conversely, p27Kip1 mRNA is down-regulated, as previously reported. In this paper we describe the activity of a peptide nucleic acid (PNA) targeting miR-221, associated to breast cancer. In order to alter the biological functions of miR-221, a polyarginine-PNA conjugate (R8-PNA-a221) was designed. R8-PNA-a221 (Arg8-AAACCCAGCAGACAATGT-NH2) showed both efficient uptake within MDA-MB-231 cell line, and it bind to the target RNA strand without 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 (measured by RT-qPCR) and induced up-regulation of a known target of miR-221, the onco-suppressor p27Kip1, mRNA and protein, measured by RT-qPCR and Western Blot analysis. The major conclusion of this manuscript is that efficient delivery of anti-miR PNA through a suitable peptide carrier (R8-PNA-a221) 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 human breast cancer: uptake and modulation of miR-221 biological functions.
BROGNARA, Eleonora;FABBRI, Enrica;MONTAGNER, Giulia;BIANCHI, Nicoletta;FINOTTI, Alessia;BREVEGLIERI, Giulia;BORGATTI, Monica;GAMBARI, Roberto
2013
Abstract
MicroRNAs (miRNAs) are an abundant class of naturally occurring, small non-coding RNA molecules that measure ~19-25 nucleotides in length able to regulate gene expression at the post-transcriptional level by (i) cleavage of target mRNAs to induce their degradation or alternatively by (ii) translational repression of protein-coding genes, depending on the degree of complementarity between miRNAs and the target sequences. In cancer, miRNAs play a double role: miRs exhibiting tumor-suppressor properties usually target mRNAs coding oncoproteins, while cancer-promoting miRs (onco-miRNAs) target mRNA coding for tumor-suppressor proteins. The oncomiR miR-221 is highly expressed in human breast cancer. MDA-MB-231 and MCF-7 cells were first characterized with respect to expression of miR-221 and p27Kip1. Our experiments (done by RT-qPCR) confirm that miR-221 is up-regulated in MDA-MB-231 cells in respect to MCF-7 cells and, conversely, p27Kip1 mRNA is down-regulated, as previously reported. In this paper we describe the activity of a peptide nucleic acid (PNA) targeting miR-221, associated to breast cancer. In order to alter the biological functions of miR-221, a polyarginine-PNA conjugate (R8-PNA-a221) was designed. R8-PNA-a221 (Arg8-AAACCCAGCAGACAATGT-NH2) showed both efficient uptake within MDA-MB-231 cell line, and it bind to the target RNA strand without 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 (measured by RT-qPCR) and induced up-regulation of a known target of miR-221, the onco-suppressor p27Kip1, mRNA and protein, measured by RT-qPCR and Western Blot analysis. The major conclusion of this manuscript is that efficient delivery of anti-miR PNA through a suitable peptide carrier (R8-PNA-a221) 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.
