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Roy Joseph, Om P. Srivastava, Roswell R. Pfister; Down-Regulation of β-Actin Gene in Human Keratoconus Corneas is due to a Stabilizing Factor Human Antigen R (HuR) Protein. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1090.
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The purpose of this study was to determine the reason for the down-regulation of β-actin gene in the stroma of human keratoconus compared to 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. The primers were designed using Primer3 for β-actin, human antigen R (HUR), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR products were analyzed using agarose gel electrophoresis. The RNA extracted from stroma of both normal and keratoconus corneas were reverse transcribed to cDNA for quantitative real-time PCR using Bio-Rad IQ5 real time thermocycler with SyBr Green.
The expression of β-actin gene was down-regulated in the stroma of the three keratoconus corneas but not in the stroma of normal and Fuch’s dystrophic corneas. The real-time PCR analysis of HuR gene showed a 6-fold difference in its expression in keratoconus corneas, which also exhibited decreased expression of β-actin gene. The GAPDH gene expression was used as an internal standard in all the above analyses, which remained at the same levels in the stroma of normal and keratoconus corneas.
It is known that β-actin mRNA has long half life and HuR binding to U-rich element is involved in its mRNA stability. Therefore based on the above results, down-regulation of β-actin gene during keratoconus could be due to a decrease in HuR protein expression.
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