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Judy Lynn Chen, Benjamin Ray Lin, Ricardo F Frausto, Andres Codriansky, Doug Chung, Anthony J Aldave; Elucidating the molecular basis of PPCD3: effect of reduced ZEB1 levels on corneal endothelial cell migration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1182.
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© ARVO (1962-2015); The Authors (2016-present)
Posterior polymorphous corneal dystrophy 3 (PPCD3) is characterized by a multilaminar appearance of the typically unilaminar corneal endothelial cells and is associated with heterozygous truncating mutations in ZEB1. The purpose of this study is to determine the effects of reduced ZEB1 protein on human corneal endothelial cell (HCEnC) migration.
HCEnC migration was measured using a non-traumatic wound-healing assay using a HCEnC line (HCEnC-21T). Cells were seeded in a 12-well plate within two-welled silicone inserts that established a 500±50 μm gap. Cells were transfected with siRNA targeting ZEB1 (ZEB1 siRNA) or scrambled siRNA (control siRNA). As previous experiments demonstrated maximum ZEB1 knockdown at 48 hours, migration experiments commenced at this time point. Forty-eight hours post-transfection, the inserts were removed, and cells were imaged hourly for 15 hours. Migration rates were calculated using ImageJ software. Data was graphed and statistical analysis was performed using GraphPad Prism software.
At 15 hours HCEnC transfected with ZEB1 siRNA had migrated 472 μm (31.4 μm/hr) compared with cells transfected with control siRNA, which had migrated 471 μm (31.5 μm/hr). Additional analysis revealed that the early-phase (0-4 hours) migration rate of ZEB1 siRNA-transfected cells (26.1 μm/hr) was 21% slower than control siRNA-transfected cells (33.2 μm/hr). Conversely, the late-phase (4-16 hours) migration rate of ZEB1 siRNA-transfected cells was 8% faster (33.3 μm/hr vs. 30.9 μm/hr). For the ZEB1 siRNA-transfected cells, the late-phase migration rate was 23% faster than the early-phase migration rate.
Although PPCD3 is associated with the appearance of multilaminar endothelial cells, the role of altered ZEB1 expression in the development of this abnormality remains to be elucidated. Although reduced ZEB1 expression does not affect the average hourly migration rate of HCEnC, two distinct migration phases were identified in HCEnC with ZEB1 knockdown. An initial reduced migration rate was followed by an increase in migration rate, the latter possibly reflecting the induction of a compensatory mechanism to establish normal migratory properties to endothelial cells in the setting of reduced ZEB1 levels.
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