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Larry Estlack, Kurt J. Schuster, Katharine E. Sheldon, Brent J. Lavey, Robert J. Thomas; siRNA-based Suppression Of VEGFC Expression In a Human RPE Cell Line Does Not Elicit Protection From Exposure To a 2- µM Laser. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1390.
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To study the effect of RNA interference (RNAi) on the expression of VEGFC in RPE cells in association with exposure to a 2-µm laser.
Introduction of synthetic short interfering RNA (siRNA) can induce RNA interference in mammalian cells. A human retinal pigment epithelial cell line (hTERT-RPE1) was transfected with RNAi target selection plasmids for VEGFC. The cell line was characterized to determine the relative abundance of VEGFC protein and mRNA levels. A preconditioning heating regime was used to evaluate VEGFC induction due to thermal stress. The RPE VEGFC knockout cells were exposed to 2-µm laser radiation at different irradiances to determine the threshold damage irradiance (ED50). The ED50 values for the RPE knockout cells were then compared to ED50 values of non-transfected control cells.
siRNAs are 21-25 bp dsRNA with 3’ overhangs that are processed from longer dsRNA by DICER in the RNA interference pathway. Vector-based siRNA approach was used to produce stable gene silencing in RPE cells. The VEGFC knockout cell lines showed a greater than 90% reduction in protein and mRNA expression. However, there was no significant increase in threshold damage irradiance (ED50) in the VEGFC knockout cells (22 W/cm2) versus control (25 W/cm2).
Using the mechanism of RNAi to silence the expression of the target gene for VEGFC in RPE cells does not elicit a protection pathway for cell survival from exposure to a 2-µm laser, or similar thermal stimuli. The results would indicate that VEGFC is not the sole factor responsible for cellular response to thermal stress, but may still be involved in the signaling pathway.
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