Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells

Abstract

Abstract. The activity of a peptide nucleic acid (PNA) targeting cancer-associated microRNA-221 is described. PNAs against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in the cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-PNA-a221) showed both very high affinity for RNA and efficient cellular uptake without the addition 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 i) lowering of the hybridization levels of miR-221 measured by RT-qPCR, ii) upregulation of p27 Kip1 gene expression, measured by RT-qPCR and western blot analysis. The major conclusion of this study 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.

Peptides for nucleic acid delivery

“…This was for example shown in mouse models of severe combined immunodeficiency (SCID) and for genetic targeting in haematopoietic progenitor cells in mice [40,41]. More recently, PPNAs demonstrated promising results as antimiRs, in vitro targeting mir-221 in breast cancer MDA cell lines and targeting mir-155 in lymphoma mouse models [42,43]. This has renewed the interest in this chemistry as a promising therapeutic modality.…”

Prognostic and biological significance of microRNA-221 in breast cancer

“…Breast cancer (BC) is considered the top cancer in women in both the developed and the developing countries.BC is a heterogeneous disease regarding molecular alteration and clinical outcome 12 . TNM staging is considered the standard method in routine use for identifying BC prognosis, for the purpose of identifying patients of BC high risk mortality, and also who could respond to treatment 3 .Recently, molecular techniques, especially gene expression profiling, have been used increasingly, in order to improve BC classification and to determine patient prognosis and response to therapy 4 .…”

MicroRNAs in breast cancer: oncogene and tumor suppressors with clinical potential

“…To achieve loss-of-function in microRNAs, 2′-O-methyl oligonucleotides (Meister et al, 2004), antagomirs (intravenous administration of cholesterol-conjugated AMOs) (Krützfeldt et al, 2005), locked nucleic acid (LNA)-oligonucleotides (Ørom et al, 2006), and microRNA sponges (microRNA inhibitory transgenes) (Ebert et al, 2007) have been employed to inhibit exogenously introduced microRNAs with high specificity. Also, active small molecule clinical compounds and peptide nucleic acids (PNAs) (Brognara et al, 2012), such as 'azobenzene' , have been tested for their ability to inhibit the expression of specific microRNAs. To improve cellular delivery, the methods for short-interfering RNA (siRNA) or short heteroduplex RNA (shRNA) could also be applied to microRNAs (Dykxhoorn et al, 2006), although excessive shRNA may increase the probability of off-target silencing and elicit nonspecific effects.…”