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Jason Rodier, Ajay Sharma, Ashish Tandon, Audra Stallard, Rajiv Mohan; RNAi Gene Silencing Of TGF-beta Signaling: A Powerful Approach To Control Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5241.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor β (TGFβ) promotes keratocyte transdifferentiation to myofibroblast and cause corneal fibrosis in vivo. Our siRNA studies demonstrated that TGFβ primarily uses SMAD signaling for this transformation in the cornea. The present study tested the hypothesis that SMAD-2, SMAD-3 or SMAD-4 gene silencing is a novel approach to treat corneal scarring using an in vitro model. We quantified the potency of SMAD gene silencing to abrogate TGFβ pathology and myofibroblast formation using SMADs siRNA, and (2) examined whether RNA inference (RNAi) or short-hairpin RNA (shRNA) is a better modality to achieve sustained SMAD gene silencing in the cornea for gene therapy using an in vitro model.
: Human donor corneas were used to obtain human cornale fibroblasts (HCF). TGFβ1 (5ng/ml) was used to induce HCF transformation myofibroblasts under serum-free conditions. Commercial pre-validated siRNA specific for SMADs were used. The shRNA and RNAi sequences specific for SMAD-2, SMAD-3 and SMAD-4 were designed using RNAi software, and cloned into mammalian expression vectors to generated SMAD-shRNA/RNAi vector constructs. Lipofectamine-2000 was used for transfection. Quantitative real-time PCR, western blotting and immunohistochemistry measured the levels of profibrotic genes [alpha smooth muscle actin (SMA), f-actin, tenascin, collagens] in SMAD-transfected and un-transfected samples.
HCF exposed to TGFβ1 showed significant SMA+ cells (80-90%, p<0.001). Significantly increased phosphoSMAD-2, phosphoSMAD-3 and SMAD-4 nuclear translocation (p <0.01-0.05) was detected without change in total SMAD. All tested gene-silencing modalities (siRNA, miRNA and shRNA) significantly blocked SMAD-2, SMAD-3, and SMAD-4 in HCF (72-90%; p<0.01). The siRNA provided transient SAMDs silencing and confirmed that TGFβ1 uses SMAD to generate myofibroblasts. The shRNA or RNAi demonstrated sustained SMADs inhibition and SMA mRNA and protein decrease (up to 95%, p<0.01). Analysis to compare gene-silencing efficiencies of shRNA and RNAi and other profibrotic genes is underway.
SMAD-2, SMAD-3 or SMAD-4 gene silencing with shRNA or RNAi can be used to treat corneal scarring. In vivo studies are warranted.
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