Over-Expression of OPTN Micro-RNA Results in Stable Knock-Down of Optineurin Protein in RGC-5 Cells

Abstract

Purpose: : Protein expression and function are tightly regulated by numerous cellular processes. With the discovery of gene-silencing small RNA molecules and the advances in bioinformatics, a prominent role has been identified for endogenously expressed small RNA molecules called micro-RNAs (miRNAs) in the post-transcriptional regulation of genetic material. The purpose of these experiments was to determine the effect of stably over-expressed OPTN miRNAs on RGC-5 cells.

Methods: : OPTN-specific miRNA sequences (Invitrogen) were selected and cloned into pcDNA^TM 6.2-GW/EmGFP expression vectors as per manufacturer’s instructions. RGC-5 cells were cultured in DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin. Lipofectamine 2000 was used to transfect RGC-5 cells with the constructed expression vectors. 48 hours post-transfection, cells were transferred to larger culture vessels for selection with growth media containing 10 µg/mL Blasticidin (Bioshop Canada). Fresh Blasticidin-containing media was replaced every 3-4 days. Within one week, resistant colonies were picked and individual clones were expanded. For assays, cells were differentiated for 24 hours with 316 nM staurosporine to express neuronal characteristics of the RGC-5 cells. Transfection efficiency was measured based on fluorescence of the GFP in the expression vectors. Knockdown of optineurin was measured using immunohistochemistry and western immunoblot analysis.

Results: : Over-expression of OPTN miRNA in RGC-5 cells resulted in significant reduction in the expression level of optineurin protein as soon as 24 hours post-transfection and lasted as long as 96 hours post-transfection. OPTN miRNA could be stably expressed in RGC-5 cells up to four passages (1:10 split). Subsequent passages resulted in a recurrence of protein expression eventually returning to 100 % expression by passage 8.

Conclusions: : Bioinformatic miRNA profiling has identified approximately 1000 miRNAs in the humane genome that play an important role in the post-transcriptional regulation of protein expression. In these experiments we show that over-expression of a miRNA targeting the OPTN gene results in a significant reduction in the expression of the optineurin protein. Moreover, this miRNA could be stably expressed in the RGC-5 cells line, thereby providing a powerful tool in studying the role of this protein in the survival of retinal ganglion cells.

Keywords: ganglion cells • gene/expression • retinal degenerations: cell biology