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Sriniwas Sriram, Gregory S. Schultz, Alfred Lewin, Paulette M. Robinson, Daniel J. Gibson, Sonal S. Tuli; Combination siRNA Treatment to Knockdown CTGF, TGF-ß1, TGF-ßR2 and Collagen in Rabbit Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2209.
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Corneal scarring remains a serious complication that can ultimately lead to functional vision loss. It is regulated by transforming growth factor ß1 (TGF-ß1), which binds to the TGFß receptor (TGF-ßR2) and induces their downstream mediator, connective tissue growth factor (CTGF). The purpose of this study was to determine the efficacy of a combination siRNA treatment in targeting these three key regulators of corneal scarring.
Short interfering RNA (siRNA) sequences were transfected into cultured rabbit corneal fibroblasts using Mirus transfection reagent. Levels of mRNA and protein for the three target genes, collagen, and α- smooth muscle actin were measured using ELISAs and qRT-PCR. Due to the interdependent action of the growth factors, it was hypothesized that a combination of the most potent mRNAs for the 3 targets was the most effective treatment to reduce the fibrotic response. Accordingly, single, double and triple siRNA combinations were tested.
Three to five different siRNAs for each target gene were tested and two with ≥80% reduction in protein and mRNA levels were identified for each gene. Combinations of the most effective siRNAs for two genes were then tested for gene knockdown. For example, TGF-ß1 siRNA-#1 and CTGF siRNA-#2 at a combined concentration of 30nM (15 nM each) gave a TGF-ß1 knockdown of ~62% and a CTGF knockdown of ~79%, compared with knockdown of ~30% and 39%, respectively, by the individual siRNAs. The degree of synergism among the different siRNA combinations was quantified using a numerical parameter called the Combined Index (CI) value. The best triple combination of siRNAs at a combined concentration of 30 nM (10 nM each) each gave 83.1% collagen knockdown compared to a scrambled control, compared with 62.8%, 70.4% and 42% knockdown by 30nM of each individual TGF-ß1, CTGF and TGF-ßR2 siRNAs, respectively. At 60nM total concentration, the triple combination gave 97% knockdown of collagen mRNA
The results demonstrate that a triple combination of siRNAs targeting TGF-ß1, TGF-ßR2, and CTGF knocked down expression of collagen type 1 mRNA more effectively than single or double combination of siRNAs.
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