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Siddharth Anil Mahajan, Marko Pastak, Olga Rotan, Matthias Epple, Friedrich E Kruse, Thomas Armin Fuchsluger; Protection of corneal endothelial cells by silencing of pro-apoptotic Bax using nano-particle mediated RNA transfer. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1178.
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© ARVO (1962-2015); The Authors (2016-present)
To protect corneal endothelial cells by silencing the proapoptotic Bax gene using biodegradable calcium phosphate nanoparticles. We hypothesize that silencing of Bax enhances survival of corneal endothelial cells under specific apoptotic stimuli.
Triple shell calcium phosphate nanoparticles with siRNA against Bax were optimized for human corneal endothelial cell (EC) transfection using HCEC-12 cell line. Experiments were performed to obtain the optimum level of transfection using different concentrations of CaP-siRNA-NPs. The setup showing best balance between transfection efficiency and cell viability was used for further experiments. Etoposide was used for induction of apoptosis which was then analyzed by Annexin V / Propidium Iodide apoptosis assay using quantitative flow cytometry. Following treatment the cells were lysed with lysis buffer and lysates were analyzed for levels of Bax and beta-actin (for normalization) protein expression. Protein expression was analyzed using ELISA and Western Blotting. Post transfection viability of cells was assessed by performing MTT cell viability assay.
Apoptosis was induced in EC using different concentrations of Etoposide (10, 25, 50, 100 and 250 µg/mL) for specified time intervals (0, 2, 4, 6, 8, 10 and 12 hours). With etoposide concentration of 100µg/mL a 3-fold increase in Bax levels was observed after 8hr of apoptosis induction. Transfection of EC with different concentrations (0.5, 1, 2.5, 5, 10 and 20nM) of CaP-siRNA-NP was performed. In the presence of the apoptotic stimulus, transfected EC (with 10nM siRNA) showed decreased level of Bax protein. Flow cytometry data reveals that EC treated with siRNA show upto 4-fold decrease in apoptotic populations as compared to the untreated samples.
We demonstrate that silencing of pro-apoptotic Bax leads to reduced cell death in EC. This strategy could be transferred to an eye bank setting to protect EC of corneas during storage.
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