In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3–1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3′-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3′-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3–1, CuFi-1, and NuLi-1 cells by pre–miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB–dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.

The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR‑221 and miR‑222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR‑221 and anti-miR‑222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222.

The role of the microRNA miR-93-5p on the secretome profile and the expression levels of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) was investigated in the neuroblastoma SK-N-AS cell line by Bio-Plex analysis and RT-qPCR. The results indicate that VEGF and IL-8 are the major miR-93-5p molecular targets. This conclusion was based on in vitro transfection with pre-miR-93-5p and anti-miR-93-5p; these treatments inversely modulated both VEGF and IL-8 gene expression and protein release in the neuroblastoma SK-N-AS cell line. Computational analysis showed the presence of miR-93-5p consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93-5p and confirming a potential regulatory role of this microRNA.