Pro-fibrotic role of miR-21 and its modulation in Precision Cut Lung Slices model

Recent studies have demonstrated that elevated expression of miR-21 may play a relevant role in the development of fibrosis by promoting the proliferation of interstitial fibroblasts and increasing the abnormal deposition of the extracellular matrix. We aimed to evaluate the involvement of miR-21 in Epithelial to mesenchymal transition (EMT) model of whole lung tissue obtained by human Precision Cut Lung Slices (PCLS). PCLS generated from tumor-free tissue of lung cancer resection surgeries were cultured in presence of Neutrophil Extracellular trap (NETs) and TGF-beta + platelet lysate (FC). PCLS were treated with miR-21 inhibitor or a Negative Control (NC). Fibrotic markers (α-SMA, Col 1 and Col 3) and miR-21 expression levels were evaluated by RT-PCR. MiR-21 relative expression was up-regulated using different stimuli (15-fold with FC and 18-fold with NETs compared to untreated). Otherwise, its expression levels resulted down-regulated (44%) by miR-21 inhibitor already after 24 hours compared to NC and untreated-PCLS. PCLS were also generated from fibrotic lung explant, in which miR-21 inhibition reduced miR-21 expression from 24h to 72h. Furthermore, miR-21 s expression level correlated with fibrotic markers (collagen1 and 3 and alpha-SMA). In particular, down-regulation of Col1 and Col3 were found significantly affected after only 24 hours of culture with miR-21 inhibition (51% and 49% respectively compared to NC and untreated) while α-SMA expression levels were found reduced at 24h to 48h (45% and 49% respectively). Inhibition of miR-21 decreases the expression of EMT markers on PCLS suggesting that this miRNAs could represent a therapeutic target as already suggested in other clinical setting.