Abstract
Purpose :
Previous studies showed that MDM2 G309 is a risk factor for proliferative vitreoretinopathy (PVR), and G309 could enhance vitreous-induced Mdm2 expression in vitro. Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of PVR. Thereby, we aimed to demonstrate the role of Mdm2 in transforming growth factor (TGF)-β2-induced EMT in retinal pigment epithelial cells.
Methods :
Inactive dead Cas9 (dCas9) was generated from a modified lenticrispr v2 (Cat. #52961, Addgene) with two mutations (D10A and H840A). An MDM2-sgRNA was targeted to the second promoter of MDM2 for blocking TGF-β2-induced expression in ARPE-19 cells. The cloning of MDM2-sgRNA to express dCas9 was confirmed by Sanger DNA sequencing. The lentivirus containing MDM2-sgRNA/dCas9 or lacZ-sgRNA/dCas9 was used to infect ARPE-19 cells, respectively. EMT was induced by TGF-β2 and identified by immunofluorescence and western blot analysis. The used markers of EMT included α-Fibronectin, N-cadherin and Vimentin. Finally wound-healing and proliferation assays were performed for testing the role of Mdm2 in these cellular responses induced by TGF-β2.
Results :
TGF-β2 is elevated in the vitreous from PVR patients and plays a critical role in EMT. Our results showed that TGF-β2 induced Mdm2 expression in ARPE-19 cells (Mean Difference [MD] = 0.91, 95% Confidence Interval (CI): 0.35-1.47, P = 0.0011) and that dCas9 directed by the MDM2-sgRNA targeting the second promoter blocked TGF-β2-stimulated expression of Mdm2 (MD = 0.38, 95% CI: 0.07-0.68, P = 0.0175). Accordingly, we found that there was lower expression of α-Fibronectin, N-cadherin in the RPE cells with MDM2-sgRNA/dCas9 than those with lacZ-sgRNA/dCas9 (α-Fibronectin: MD = 0.90, 95% CI: 0.27-1.53, P = 0.0033; N-cadherin: MD = 0.68, 95% CI: 0.05-1.32, P = 0.03). Subsequently, our experimental data demonstrated that dCas9 directed by MDM2-sgRNA attenuated TGF-β2-dependent cell migration (MD = 0.30, 95% CI: 0.23-0.40, P < 0.0001) and proliferation (MD = 1.73, 95% CI: 0.65-2.81, P = 0.0045).
Conclusions :
The CRISPR/dCas9's capability of blocking the TGF-β2-induced expression of Mdm2 and EMT can be exploited for a potential therapeutic approach to PVR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.