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C. Yuan, E.J. Zins, A.F. Clark, A.J. W. Huang; Suppression of Myocilin Expression by Small Interfering RNAs . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3792.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations of the myocilin gene have been associated with open–angle glaucoma. The pathogenesis of myocilin–related glaucoma is likely mediated via protein misfoldings. Using transformed HEK293 and trabecular meshwork (TM) cell lines as model systems, we investigated the feasibility of applying RNA interference to suppress the myocilin expression and to ameliorate the cytotoxic effects induced by mutant myocilins. Methods: Human myocilin cDNA as the target gene was subcloned and fused with EGFP gene (MYOCpEGFP). Myocilin–specific small interfering RNAs (siRNAs) were designed by our in–house program SiFinder. An RNA polymerase III promoter–driven vector (pH1–RNA) was used to generate siRNAs in vivo. Cultured HEK293 or TM5 cell lines were co–transfected with MYOCpEGFP and these myocilin–specific siRNA–generating plasmids. Suppression of myocilin by siRNAs was evaluated by fluorescence microspcopy and Western blotting. Protein misfolding and endoplastic reticulum (ER) stress response induced by mutant myocilins were evaluated with BiP promoter–driven luciferase reporter assays. Results: Co–transfection of MYOCpEGFP and siRNA–generating plasmids targeting the myosin–like and the olfactomedin–like domains showed effective reduction of the fusion protein. Specifically, these siRNAs targeted regions containing amino acid residues R76, E352, K423 and N480 known to be associated with primary open angle glaucoma (POAG) or juvenile open angle glaucoma (JOAG). The suppression efficiencies of these siRNAs were as high as 90%. The activation of BiP gene was significantly reduced when the expression of mutant myocilin proteins was suppressed by myocilin–specific siRNA. Conclusions: The selected myocilin–specific siRNAs effectively suppressed myocilin expression and reduced the ER stress response in cultured cells.
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