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M. Duong, A.K. Ball; Sirna–Mediated Knockdown of Optineurin in Rgc–5 Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):172.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the optineurin gene (OPTN) may an important factor involved in the onset of low–pressure primary open–angle glaucoma. The purpose of these experiments is to use siRNA technology to silence the expression of OPTN and study this effect on RGC survival. This will help us elucidate the role of optineurin in normal retinal cell function.
Immunohistochemistry was performed on fixed–frozen sections of rat retina using anti–sera directed against optineurin to localize the protein within the retina. RGC–5 cells were cultured in DMEM/F12 supplemented with 10% FBS and 1g/L glucose. Cells were differentiated for 24 hours with 1 µM staurosporine before treatment. Immunocytochemistry was performed to examine expression of optineurin in RGC–5 cells. siRNAs directed against OPTN mRNA were sequenced by The Whitehead Biomedical Research Institute. The siRNAs were scanned using the NCBI BLAST algorithm to exclude siRNAs with homologies to other genes in the rat genome, other than the target gene. siRNAs were synthesized using the SilencerTM siRNA Construction Kit. Lipofectamine 2000 was used to transfect RGC–5 cells with siRNAs directed against OPTN. Verification of transfection efficiency was determined in control transfections of RGC–5 cells using Cy–3 labeled siRNAs directed against luciferase. siRNA–mediated knockdown of optineurin was measured using western immunoblot analysis at 4 hours, 8 hours and 24 hours, post–transfection.
Optineurin immunoreactivity was localized to the GCL, the OPL and the RPE. Optineurin immunopositive cells were double–labeled with anti–sera directed against PGP 9.5 and Thy 1, demonstrating that cells expressing optineurin in the GCL were RGCs. Western immunoblot analysis showed that siRNAs directed against optineurin were capable of specifically reducing the expression of optineurin in RGC–5 cells in a time dependent manner. Transfection durations longer than 24 hours resulted in cell death of RGCs.
We have localized optineurin expression in the rat retina to the RGCs, the OPL and cells in the RPE. We have also shown optineurin expression in RGC–5 cells in culture. siRNA–mediated reduction of optineurin in RGC–5 cells is time–dependent within 24 hours. Following 24 hours, optineurin knockdown results in cell death. These results provide evidence for an important role for optineurin expression on RGC survival and implicates it as a potential target for therapeutic intervention in certain forms of glaucoma.
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