D2.178 - Evaluation of Therapeutic Potential of The miRNAs In Asthma via Transfection of Airway Epithelial Cells with Agomir/Antagomir Loaded Exosomes

Recent studies have shown that miRNAs play an important role in the pathogenesis of asthma and they have the potential to be used as biomarkers in asthma or as an alternative new treatment approach by rearranging the expression of genes that play a role in asthma. In this project, it was aimed to suppress the pathways related to asthma pathogenesis in which certain miRNAs, known to be expressed in lung epithelial cells with increased or decreased expression in asthma, by transferring inhibitors (antogomirs) or mimics (agomirs) to epithelial cells.

Based on a literature review, four different miRNAs were selected that are known to affect the expression of epithelial-released mediators, including IL-1β, TGF-β, IL-6, IL-8, Eotaxin-1, VCAM, VEGF, MMP9, TIMP-1, and GM-CSF, and that exhibited either an increase (miR-19a and miR-155) or a decrease (miR-133a and miR-181b) in asthma pathogenesis.  first naturally produced exosomes were isolated from lung epithelial cells, Calu-3 cells, and characterized using flow cytometry, a nanoparticle tracking analyzer, and scanning electron microscopy. Subsequently, fluorescently labeled agomir and antagomir of the selected miRNAs were transfected into exosomes and transferred to Calu-3 cells, and transfection was confirmed by examination under a fluorescence microscope. Whether the loaded exosomes were taken into the cells also demonstrated using RT-PCR. The toxicity of the applied agomir and antagomir doses to Calu-3 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay. After incubating exosomes loaded with the determined dose of unlabeled miRNA with Calu-3 cells for 24 hours, changes in gene expression of mediators that are targets of the selected miRNAs were determined using RT-PCR and ELISA. 

RT-PCR experiments revealed that exosome-mediated delivery of miR133a agomir to Calu-3 cells suppressed GM-CSF and MMP9 expression. When miRNA 181b agomir was transferred to Calu-3 cells, we observed suppression of VCAM and IL-6 expression, which are among the target genes of this miRNA. As expected, we observed an increase in VEGF expression after stimulating cells with miR19a agomir, which has been shown to be increased in asthma, while the addition of antagomir suppressed it. 

Our data support the potential use of agomir and antagomir as an alternative treatment modality for asthma.