MicroRNAs (miRNAs, miRs) are a family of small (19 to 25 nucleotides in length) noncoding RNAs that regulate gene expression by sequence-selective targeting of mRNAs (1-4), leading to a translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and the target sequences (2). Since a single miRNA can target several mRNAs and a single mRNA may contain several signals for miRNA recognition, it is calculated that at least 10-40% of human mRNAs are targets of microRNAs (2). In general, a low expression of a given miRNA is expected to be potentially linked with an accumulation of targets mRNAs; conversely, a high expression of miRNAs is expected to be the cause of a low expression of the target mRNAs. In this paper we describe the activity of a peptide nucleic acid (PNA) targeting micro-RNA 221, associated to breast cancer. PNA against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in target cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-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 Rpep-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by 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 Blot analysis. The major conclusion of this manuscript is that efficient delivery of anti-miR PNA through a suitable peptide carrier (Rpep-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 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells
BROGNARA, Eleonora;FABBRI, Enrica;BIANCHI, Nicoletta;FINOTTI, Alessia;BREVEGLIERI, Giulia;BORGATTI, Monica;GAMBARI, Roberto
2012
Abstract
MicroRNAs (miRNAs, miRs) are a family of small (19 to 25 nucleotides in length) noncoding RNAs that regulate gene expression by sequence-selective targeting of mRNAs (1-4), leading to a translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and the target sequences (2). Since a single miRNA can target several mRNAs and a single mRNA may contain several signals for miRNA recognition, it is calculated that at least 10-40% of human mRNAs are targets of microRNAs (2). In general, a low expression of a given miRNA is expected to be potentially linked with an accumulation of targets mRNAs; conversely, a high expression of miRNAs is expected to be the cause of a low expression of the target mRNAs. In this paper we describe the activity of a peptide nucleic acid (PNA) targeting micro-RNA 221, associated to breast cancer. PNA against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in target cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-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 Rpep-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by 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 Blot analysis. The major conclusion of this manuscript is that efficient delivery of anti-miR PNA through a suitable peptide carrier (Rpep-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.
