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Benjamin Ray Lin, Ricardo F Frausto, Judy Lynn Chen, Doug Chung, Anthony J Aldave; Elucidating the molecular basis of PPCD3: reduced ZEB1 protein levels do not affect corneal endothelial cell apoptosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4903. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Posterior polymorphous corneal dystrophy 3 (PPCD3) is associated with a monoallelic deficiency secondary to nonsense and frameshift mutations in ZEB1. The purpose of this study was to investigate the impact of ZEB1 knockdown on the induction of apoptosis via doxorubicin hydrochloride (DOX) in a human corneal endothelial cell line.
ZEB1 protein levels in a human corneal endothelial cell line (HCEnC-21T) were reduced by transfection with 4 nM ZEB1 siRNA, with scrambled siRNA used as a control. Forty eight hours after siRNA transfection, HCEnC-21T cells were treated with 2.5µM of DOX. Whole-cell protein lysates were prepared at 0, 3, 6, 9, and 12 hours after DOX treatment and subjected to SDS-PAGE. Apoptosis was monitored using phase-contrast microscopy. ZEB1 and cleaved caspase 3 (cCASP3), an apoptosis marker, were detected by immunoblotting. Densitometric analysis for ZEB1 and cCASP3 protein levels was performed using ImageJ software.
Forty-eight hours after transfection with ZEB1 siRNA, ZEB1 protein levels were reduced by 58% while no reduction was observed after transfection with scrambled siRNA. Phase-contrast microscopy demonstrated an increasing number of phase-positive cells and other features indicative of apoptosis in both ZEB1 siRNA and scrambled siRNA transfected cells as DOX treatment time increased. Transfection with ZEB1 siRNA did not significantly impact the induction or progression of apoptosis as measured by the appearance of cCASP3 protein 12 hours after DOX treatment. ZEB1 protein levels were inversely correlated with increasing cCASP3 protein levels. Twelve hours after DOX treatment, a significant decrease was observed in the levels of ZEB1 from levels 0-3 hours after DOX treatment in both ZEB1 siRNA and control siRNA transfected cells.
A ZEB1 protein deficiency secondary to truncating mutations in ZEB1 is postulated as the cause of PPCD3. Reduction of ZEB1 protein levels in HCEnC-21T cells does not impact the induction or progression of apoptosis. However, an inverse relationship between ZEB1 levels and cCASP3 was observed, suggesting that ZEB1 levels are negatively regulated downstream of apoptotic stimuli. Although an initial reduction in ZEB1 levels does not have an impact on apoptosis, its reduction in response to apoptosis suggests that its role in this cellular process warrants further investigation.
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