Modulation of Epithelial Mesenchymal Transition (EMT) Through the Notch Signaling Pathway in Human Retinal Pigment Epithelial Cells

摘要

Purpose : Epithelial mesenchymal transition (EMT) of the RPE plays a central role in the development of proliferative vitreoretinopathy (PVR), a blinding clinical complication of retinal detachment and trauma. The Notch signaling pathway has been shown to regulate the expression of EMT proteins; however, its role in PVR is unknown. We examined the impact of Notch signaling in the RPE using a genome editing CRISPR/cas9 approach to knock out Notch 2/3 expression in ARPE19 cells and evaluated its impact on EMT protein expression and cell phenotype. Methods : Two gRNAs were cloned into lentiviral CRISPR/Cas9 vectors and used to target the Notch1-4 receptors. Lentiviruses were packaged in HEK293FT cells and ARPE19 cells were transduced with lentiviral Notch KO constructs. Transfected cells were isolated using puromycin and KO status confirmed by sequencing. EMT protein markers were evaluated in the Notch KO cell lines by Western blotting, and the cell phenotype was examined including proliferation, migration, and susceptibility to apoptosis. Results : Disruption of Notch2 in ARPE19 cells leads to the upregulation of N-cadherin, ?2-catenin, Snai1, ?±-SMA and downregulation of the E-cadherin and ZO-1 compared to the CRISPR/Cas9 empty vector infected controls. Knockout of Notch2 promotes ARPE19 cell migration. Conclusions : Disruption of the Notch2 gene indirectly promotes EMT-associated gene expression in ARPE19 cells. Notch pathway signaling may play an important role in contributing to the PVR phenotype expressed by transformed RPE cells following retinal detachment. This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.