March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Combination siRNA Treatment to Knockdown CTGF, TGF-ß1, TGF-ßR2 and Collagen in Rabbit Corneal Fibroblasts
Author Affiliations & Notes
  • Sriniwas Sriram
    Biomedical Engineering,
    University of Florida, Gainesville, Florida
  • Gregory S. Schultz
    Dept of OBGYN and Ophthalmology,
    University of Florida, Gainesville, Florida
  • Alfred Lewin
    Molecular Genetics and Microbiology,
    University of Florida, Gainesville, Florida
  • Paulette M. Robinson
    OB-GYN,
    University of Florida, Gainesville, Florida
  • Daniel J. Gibson
    Biochemistry & Molecular Biology,
    University of Florida, Gainesville, Florida
  • Sonal S. Tuli
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  Sriniwas Sriram, None; Gregory S. Schultz, None; Alfred Lewin, None; Paulette M. Robinson, None; Daniel J. Gibson, None; Sonal S. Tuli, None
  • Footnotes
    Support  US Department of Defense W81XWH-10-1-0917
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 2209. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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

Conclusions: : 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.

Keywords: wound healing 
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