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V. S. Yellore, S. A. Rayner, A. J. Aldave; Induction of Extracellular Keratoepithelin Expression by TGFβ1 and Inhibition of Expression by RNA Interference in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1713. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To report the increased production of extracellular keratoepithelin (KE) by corneal epithelial cells following induction by extraneously supplied TGFβ1 and the inhibition of KE production by induced/uninduced corneal epithelial cells by RNA interference.
Corneal epithelial cells cultivated in a serum-free medium were treated with 0, 10 and 20 ng/ml TGFβ1 over a period of 66 - 96 hours. Medium samples were withdrawn and centrifuged before measurement of extracellular KE production by ELISA. Corneal epithelial cells were enzymatically lifted from plates and washed with serum free medium plus soybean trypsin inhibitor and then used for RNA and protein isolation using TRIzol. Quantification of the intracellular concentration of KE and mRNA transcripts for KE were performed by Western analysis and RT-PCR, respectively. Three pre-designed siRNAs targeting TGFBI (Ambion, Austin, TX) at concentrations ranging from 0 to 30 µM were admixed with the transfection reagent, siPORT NeoFX, and used to reverse transfect TGFB1-induced and non-induced corneal epithelial cells. Intracellular and extracellular concentrations of KE were measured, as were intracellular TGBFI mRNA transcript levels.
KE is expressed by corneal epithelial cells. Induction of expression with TGFB1 results in a 2 to 3-fold increase in the amount of extracellular KE produced. Two of the three predesigned siRNAs demonstrated up to 75% knockdown of extracellular KE within 48 hours of exposure in both TGFβ1-stimulated and unstimulated cells. The level of knockdown of KE production increased with siRNA concentration up to 15µM for the two active siRNAs, while higher concentrations did not demonstrate increased knockdown, even with increased transfection reagent levels. The level of extracellular KE expression was constant for the duration of the experiments in cells not exposed to either TGFB1 or anti-TGBFI siRNAs and in the cells treated with a scrambled human siRNA.
Extracellular KE expression by corneal epithelial cells is induced two-three fold following induction with TGFβ1. However, this induction can be inhibited with anti-TGFBI siRNAs, which also inhibit KE expression by non-induced corneal epithelial cells. As the dystrophic corneal deposits in the TGFBI dystrophies consist of KE derived from the corneal epithelium, and accelerated deposit formation is thought to be mediated by TGFβ1, RNA interference represents a potential means to inhibit dystrophic deposit formation and recurrence following surgical intervention.
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