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Siddharth Mahajan, Daniel Thieme, Friedrich E Kruse, Thomas Armin Fuchsluger; Successful reduction of pro-apoptotic Bax in corneal endothelial cells using non-viral RNA transfer. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1404.
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Prevention of corneal endothelial cell (EC) apoptosis would be a functional approach to increase EC quantity and quality in corneas during storage and after transplantation. The objective of this study was to both assess the expression of pro-apoptotic marker protein Bax in EC and to optimize siRNA transfection parameters for its silencing.
The expression of Bax protein was analyzed in corneal endothelial cells following apoptosis induction with different concentrations of staurosporine (0.1 to 2.5µM). Protein expression was detected by western blotting and quantified with ImageJ. In order to investigate the effect of pro-apoptotic Bax knockdown, optimization of parameters for transfection of HCEC-12 cells with siRNA was performed. Fluorophore-conjugated control siRNA was used as it enabled us to analyze the transfection efficiency by flow cytometry. Forward and reverse transfection procedures were compared in order to find which procedure resulted in better transfection efficiency. Two different lipid-based transfection reagents were used (X-treme gene and RNAiMAX). Different concentrations of Bax siRNA (5 to 100nM) were used for transfection, then protein expression was analyzed at different time intervals (24, 48, 72hr) by western blotting.
Higher transfection efficiency was observed with Xtreme gene reagent 72 ± 9% as against 60 ± 6% for RNAiMAX reagent. Interestingly, transfection efficiency was significantly higher in reverse transfection 72 ± 9% as compared to forward transfection 45 ± 10% (p<0.005) for Xtreme gene reagent. Based on the optimized parameters, Bax siRNA transfection showed gradual decrease in Bax expression across the concentration range (20 to 100nm). Bax expression was decreased up to 15 % with lower concentrations (up to 40nM) of siRNA. However, the higher concentration of 100nM Bax siRNA resulted in significantly higher Bax knockdown of 57 ± 6% as compared to the control siRNA (p< 0.05). This decrease in Bax expression was significant at 24 and 48hr (p<0.05) but not at 72 hr after siRNA treatment.
We successfully demonstrated a transient reduction of pro-apoptotic Bax by non-viral siRNA transfer. Reverse transfection delivered better transfection efficiencies for this siRNA with Xtreme gene transfection reagent. These findings might open a potential treatment of EC which could be transferable to an eye bank setting.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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