Inhibiting effects of Src kinase inhibitor on TGF-β1 induced epithelial-mesenchymal transition of human RPE cells

Objective

To investigate the inhibiting effect of CGP77675 (CGP), a Src inhibitor, on epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial (RPE) cells induced by transformation growth factor-β1 (TGF-β1).

Methods

Human RPE cell line (ARPE19 cells) was cultured in vitro and divided into control group, TGF-β1 group and TGF-β1+ CGP group.Corresponding agent was added into culture medium based on grouping.The morphology of the cells were examined under the optical microscope 3 days after culture.The expressions of EMT-related genes and proteins in the cells were detected by real-time quantitative PCR and Western blot, respectively, including fibronectin 1 (FN1), and plasminogen activation inhibitor 1 (PAI1), and the expressions of zonula occludens protein 1 (ZO1) and cytoskeleton protein filamentous actin (F-actin) were detected by immunofluorescence staining.MTT assay was employed to evaluate the cell proliferation rate.The migration distance of the cells was measured by scratch test.

Results

The ARPE19 cells in the control group showed an epithelial-like morphology and F-actin and ZO-1 were expressed along cell membrane.In the TGF-β1 group, the cells appeared to be fibrous-like, and the fluorescence staining of F-actin was disordered and ZO-1 was discontinuous on the cell membrane.The cells in the TGF-β1+ CGP group remained to be an epithelial-like in shape with clear and complete expressions of F-actin and ZO-1.The relative expressions of FN1 mRNA and PAI1 mRNA in the cells were 0.211±0.080 and 0.116±0.073, 1.000±0.001 and 1.000±0.001, 0.368±0.097 and 0.362±0.048 in the control group, TGF-β1 group and TGF-β1+ CGP groups, showing significant differences among the groups (F=33.14, 82.92; both at P<0.01), with the highest expressions of FN1 mRNA and PAI1 mRNA in the TGF-β1 group (all at P<0.05). The relative expressions of FN1 and PAI1 proteins were 0.166±0.055 and 0.327±0.066, 1.000±0.001 and 1.000±0.001, 0.143± 0.030 and 0.260±0.077 in the control group, TGF-β1 group and TGF-β1+ CGP group, with significant differences among three groups (F=181.90, 48.85; both at P<0.01), and the expressions FN1 and PAI1 proteins were significantly higher in the TGF-β1 than those in the control group and TGF-β1+ CGP group (all at P<0.05). The cell proliferative rate in the TGF-β1+ CGP group was (79.30±3.44) % and (54.80±7.39) % at the third day and seventh day after culture, which were significantly reduced in comparison with (99.50±1.00)% and (99.10±0.50)% in the control group as well as (95.10±4.20)% and (92.10±4.50)% in the TGF-β1 group (all at P<0.05). The migration distance was disappeared in the TGF-β1 group, and the scratch width was not obviously changed in the TGF-β1+ CGP group.

Conclusions

Src inhibitor can inhibit EMT process of ARPE19 cells induced by TGF-β1, indicating that Src signaling pathway may play a critical role in EMT of RPE cells.