A DNA Tetrahedron–Delivered miR-17-5p mimic exerts dual anti-inflammatory and Anti-Pseudomonas aeruginosa effects in cystic fibrosis

Cystic Fibrosis (CF) is a genetic disease characterized by chronic pulmonary inflammation and infection, principally with Pseudomonas aeruginosa . P. aeruginosa can exhibit resistance to innate immune effectors and antibiotics by expressing virulence factors and forming antibiotic-resistant biofilms, which facilitate chronic infection in immunocompromised hosts. Therefore, new antimicrobials are required. MicroRNAs are mammalian short non-coding RNAs that negatively regulate protein expression. MiR-17-5p, an inhibitor of CXCL8/IL-8, can decrease neutrophil abundance in the CF lung. Here we investigate whether miR-17-5p has anti - P. aeruginosa properties. The sequences of the full-length miRNA and the miR-17-5p isoform miR.17.P4d_5p were tested for their ability to bind to RNA transcripts encoded by P. aeruginosa PAO1 (NC_002516) using the bioinformatics tools RocketmiR and IntaRNA. A miR-17-5p DNA tetrahedron (Td-miR.17.P4d_5p, Size = 118–180 nm) was tested for its delivery into CF bronchial epithelial cells and ability to inhibit IL-8 gene and protein expression, and its effects on P. aeruginosa growth, antimicrobial resistance, and biofilm formation. Successful delivery into CFBE41o− and 16HBE14o− cells inhibited IL-8 expression induced by Pseudomonas -conditioned medium (PAO1, Mucoid strains 1 and 2 p < 0.00001; PA14 p < 0.0001). Delivery into PAO1 and PA14 inhibited growth and biofilm formation while significantly enhancing their sensitivity to the antibiotic cefotaxime. Td-miR.17.P4d_5p also significantly inhibited biofilm formation in two clinical mucoid P. aeruginosa strains. qPCR and EMSA were used to investigate the mechanism of action of miR.17.P4d_5p. The data showed that miR.17.P4d_5p could inhibit expression of kynU ( p < 0.00001), a tryptophan metabolism pathway molecule related to PAO1 growth, and the biofilm formation-related molecules pilL , fimX , ppkA and pslA (all p < 0.00001) in P. aeruginosa PAO1. The combined anti -IL-8 and anti - P. aeruginosa properties of miR.17.P4d_5p suggest its potential usefulness as a dual anti-infective and anti-inflammatory therapeutic strategy for CF.