Abstract
Purpose: :
To determine whether siRNAs that silence RTP801 gene expression enhance RGC-5 cell survival in culture.
Methods: :
A cell line with certain ganglion cell properties (RGC-5 cells: concentration 80000/ml) in culture was subjected to a reverse transfection procedure aimed at silencing RTP801. After 48 hours the cells in culture were exposed to an insult of 500µM CoCl2 or blue light (600 lux) for 12 hours. In some instances cultures were processed for the localisation of RTP801 immunoreactivity or subjected to a dead/live staining assay. In other instances cell extracts were analysed by western blot and real-time PCR procedures to determine the expression of RTP-801.
Results: :
An insult of CoCl2 or blue light caused an up-regulation of RTP-801 protein and mRNA. Moreover, a number of dead cells were revealed by use of dead/live assay. Similar results occurred in mock-transfected cells and in the presence of non-targeted sequence siRNAs. However 50nM of a mouse siRNA (Dharmacon) or human siRNA (PF-655) caused significant silencing of CoCl2 or blue light stimulation of RTP-801 protein and mRNA. Moreover, the dead/live assay showed that the silencing of RTP-801 resulted in significantly less cells dying because of either CoCl2 or blue light insults.
Conclusions: :
Our studies support the view that reducing the production of RTP801 by gene silencing provides a potential way to protect cells from stress insults like that caused by CoCl2 or blue light.
Keywords: gene/expression • ganglion cells • hypoxia