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R. Joseph, O. P. Srivastava, R. R. Pfister; Down Regulation of β-Actin Gene in Human Keratoconus Corneas. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4969.
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The purpose of this study was to determine the expression of β-actin at transcriptional and translational levels in human keratoconus and normal corneas.
Three normal corneas (ages: 30 to 55 years) and three keratoconus corneas (ages: 30 to 55 years) were used for RNA isolation from epithelium and stroma. The β-actin gene was amplified using Access RT PCR system (Promega). The primers were designed using Primer3 for β-actin, cofilin, and GAPDH. The PCR products were analyzed using 1% agarose gel. The immunohistochemical analysis of β-actin was performed using a monoclonal β-actin antibody (Abcam). Ten µm frozen sections were used from normal, keratoconus and Fuch’s dystrophy corneas.
The β- actin gene expression was down-regulated in the stroma of the three keratoconus corneas but not in the stroma of normal and Fuch’s dystrophic corneas. However the β- actin gene expression levels were at the same levels in the epithelium of normal, keratoconus and Fuch’s dystrophic corneas. Similarly, the immunohistochemical analysis of β-actin showed that its expression was down- regulated in the stroma of keratoconus compared to the stroma of normal and Fuch’s dystrophic corneas. The expression of cofilin (a β-actin interacting protein) gene was also down-regulated in the stroma of keratoconus corneas but not in the stroma of normal and Fuch’s dystrophic corneas. However, cofilin gene expression was normal in the epithelium of normal and keratoconus corneas. The GAPDH gene expression was used as an internal standard in all the above analyses, which remained at the same levels in normal, keratoconus and Fuch’s dystrophic corneas.
β-actin and cofilin showed down-regulation at both transcriptional and translational levels in keratoconus corneas compared to normal and Fuch’s dystrophic corneas. We hypothesize that the down-regulation of β-actin gene would weaken cytoskeleton of stroma and might result in anterior protrusion of keratoconus cornea.
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