# Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19
## 2 Results
### uPAR Downregulation in Lung Epithelial Cells Induced Mesenchymal Phenotype  
We have previously shown that uPAR downregulation induced Neuro2a cell size enlargement and EMT [43]. To this end, we set out to confirm that uPAR downregulation in lung epithelial cells contributed to pulmonary fibrosis aggravation via EMT induction. In order to tackle this issue, we enrolled human A549 (adenocarcinoma human alveolar basal epithelial cells) and BEAS-2B (non-tumorigenic lung epithelial cell line) cells. We used shRNA to suppress uPAR expression (shPLAUR) and scrambled shRNA as a control. Stable cell lines expressing shPLAUR or scrambled shRNA were established after transfection and an eight-week puromycin selection. The content of uPAR at the protein level was decreased 1.5-fold and 3.6-fold in A549-shPLAUR and BEAS-2B-shPLAUR cells, respectively (Figure 8A,B).  
Cells were plated in a low-density monolayer and cultured for 24 hours to allow for cell adhesion. Live-cell images were obtained and used for cell size measurement (Figure 8C,D). uPAR downregulation resulted in a pronounced phenotypic change in A549 cells (Figure 8C) and, to a lesser extent, in BEAS-2B cells. In particular, scrambled shRNA-transfected A459 cells demonstrated an epithelial-like morphology, while shRNA A549 cells demonstrated a rounded spread shape (Figure 8C). BEAS-2B-shPLAUR cells also presented a more rounded morphology (Figure 8D). The overall size of shPLAUR-cells was significantly larger as compared to the respective controls both for A549 (\(t\)-test, ***\(p<0.0001\), Figure 8C) and BEAS-2B cells (\(t\)-test, ***\(p<0.0001\), Figure 8D).  
By using a Western blot analysis, we next examined the expression of classic markers of epithelial cell phenotype (E-cadherin) and mesenchymal phenotype (N-cadherin) in A549 and BEAS-2B cells (Figure 8E,F). The expression of E-cadherin was significantly decreased, while N-cadherin was increased in A549-shPLAUR cells (ANOVA, [PERSON]'s post hoc test, \(p<0.01\), Figure 8E), pointing to EMT induction after uPAR downregulation. Administration of uPA to the cell medium for 24 h exerted no effect on EMT marker expression: A549-shPLAUR cells persistently expressed lower levels of E-cadherin and higher levels of N-cadherin compared to A549-scrambled shRNA cells (ANOVA, [PERSON]'s post hoc test, \(p<0.05\), Figure 8E). Similar results were obtained in BEAS-2B cells: N-cadherin expression markedly increased in BEAS-2B-shPLAUR cells as compared to BEAS-2B-scrambled shRNA cells (ANOVA, [PERSON]'s post hoc test, \(p<0.001\), Figure 8F), however, E-cadherin expression remained unchanged. Along with that, uPA treatment exerted no effect on the EMT marker expression profile in BEAS-2B-shPLAUR cells treated with uPA, where cells expressed a significantly higher level of N-cadherin as compared to BEAS-2B-scrambled shRNA cells (ANOVA, [PERSON]'s post hoc test, \(p<0.05\), Figure 8F). The above results allowed us to assume that uPAR was a factor that sustained the epithelial phenotype in lung epithelial cells, while uPAR downregulation promoted the shift into a mesenchymal state with increased N-cadherin expression accompanied by substantial morphological changes.