A Peptide Nucleic Acid against MicroRNA miR-145-5p Enhances the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Calu-3 Cells

Abstract

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level.

A Peptide Nucleic Acid (PNA) Masking the miR-145-5p Binding Site of the 3′UTR of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNA Enhances CFTR Expression in Calu-3 Cells

“…Despite the fact that comparison between the anti-miR-PNA and PNA-masking approaches has not been done in parallel in this study, by comparing our results to those published by Fabbri et al [35], the PNA masking approach appears to be more effective than the anti-miR-PNA approach on the increase of CFTR. The fold increases of CFTR protein were 2-to 2.5-fold and 6-to 8-fold when the anti-miR-PNA [33] and PNA masking (Figure 4) approaches were employed, respectively, when the PNAs were used at 2 µM. Moreover, very low effects using anti-miR-145 PNA were obtained at lower concentrations (unpublished data), while the miR145-maskingPNA was active even when used at 0.5 µM.…”

“…As far as the comparison between the miRNA inhibiting and the miRNA masking strategies, we would like to underline that we have elsewhere published data supporting the use of miR-145-5p targeting in CF, based on an antisense PNA to target miR-145-5p and enhance expression of the CFTR gene [33,34]. This conclusion was recently confirmed by Kabir et al, who demonstrated that miR-145 mediates TGF-β inhibition of synthesis and function of the CFTR in CF airway epithelia [35].…”

“…The synthesis of the miR145-maskingPNA was similar to those previously reported [15,33]. The synthesis was performed using a standard Fmoc-based automatic peptide synthesizer for both the PNA and the polyArg tail.…”

“…The CFTR protein increase was found to be 6-to 8-fold in Calu-3 extracts after miR145-maskingPNA treatment in the three independent experiments in which CFTR mRNA was also analyzed ( Figure 4B, black boxes). In order to verify specificity of the effects derived by the Western blotting experiment, Calu-3 cells were cultured with a negative control PNA previously demonstrated unable to (a) interact with miR-145-5p [33] and (b) inhibit RT-qPCR amplification of CFTR mRNA sequences containing miR-145-5p binding sites (Figure 3, white symbols). The results obtained ( Figure 4B) demonstrate no increase of the relative CFTR/β-actin values in Calu-3 cells treated with the negative control PNA.…”

“…Epigenetic regulation of expression of cystic fibrosis transmembrane conductance regulator (CFTR) gene by miRNAs has been recently reported by different groups [24][25][26][27][28][29][30][31][32][33][34]. For instance, expression of miR-145 and miR-494 was found to anti-regulate CFTR [28].…”