A Peptide-Nucleic Acid Targeting miR-335-5p Enhances Expression of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene with the Possible Involvement of the CFTR Scaffolding Protein NHERF1.

Ilaria Lampronti,Roberto Corradini,Silvia Munari,Alessia Finotti,Maria Dechecchi,Alex Manicardi,Enrica Fabbri,Roberto Gambari,Jessica Gasparello,Giulio Cabrini,Tiziana Jakova,Monica Borgatti,Anna Tamanini

doi:10.3390/biomedicines9020117

Abstract

(1) Background: Up-regulation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) might be of great relevance for the development of therapeutic protocols for cystic fibrosis (CF). MicroRNAs are deeply involved in the regulation of CFTR and scaffolding proteins (such as NHERF1, NHERF2 and Ezrin). (2) Methods: Content of miRNAs and mRNAs was analyzed by RT-qPCR, while the CFTR and NHERF1 production was analyzed by Western blotting. (3) Results: The results here described show that the CFTR scaffolding protein NHERF1 can be up-regulated in bronchial epithelial Calu-3 cells by a peptide-nucleic acid (PNA) targeting miR-335-5p, predicted to bind to the 3′-UTR sequence of the NHERF1 mRNA. Treatment of Calu-3 cells with this PNA (R8-PNA-a335) causes also up-regulation of CFTR. (4) Conclusions: We propose miR-335-5p targeting as a strategy to increase CFTR. While the efficiency of PNA-based targeting of miR-335-5p should be verified as a therapeutic strategy in CF caused by stop-codon mutation of the CFTR gene, this approach might give appreciable results in CF cells carrying other mutations impairing the processing or stability of CFTR protein, supporting its application in personalized therapy for precision medicine.

Highlights

MicroRNAs are noncoding RNAs from 19 to 25 nucleotides in length that regulate gene expression by targeting mRNAs, leading to a translational repression or mRNA degradation [1,2,3]
With respect to peptide-nucleic acid (PNA)-based targeting of miRNAs involved in Cystic Fibrosis TransmembraneConductance Regulator (CFTR) regulation, we recently reported that a PNA directed against miR-145-5p inhibits its biological functions and up-regulates CFTR expression [36]
Venn of miRNAs not modulated, or up- (FC > 2) and down- (FC: 2–3; FC > 3) modulated. (C) Venn diagram showing miR-155-5p as the only miRNA in common with the three lists

Summary

Introduction

MicroRNAs (miRNAs) are noncoding RNAs from 19 to 25 nucleotides in length that regulate gene expression by targeting mRNAs, leading to a translational repression or mRNA degradation [1,2,3]. A second analysis of miRNA profiling showed high expression of miR-494 and miR-509-3p in CF cells and a direct interaction with the CFTR transcript [10]. These in vitro findings were confirmed and extended in ex vivo analyses which evidenced an increased expression of miR-494, miR-223 and miR-145 in CF brushings of airway cells [11]. Different miRNAs which have been found to be increased in CF primary bronchial epithelial cells can reduce CFTR expression, either by direct (miR-145, miR-223, miR-494, miR-509-3p) or indirect (miR-138) targeting

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Discussion

Conclusion