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Roy Joseph, Om P. Srivastava, Roswell R. Pfister; Down-Regulation of β-Actin in Keratoconus is Mediated Through Human Antigen R (HuR). Invest. Ophthalmol. Vis. Sci. 2012;53(14):1107.
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The purpose of this study was to determine if relative to normal corneas, the keratoconus corneas exhibit a down-regulation of β-actin gene in the stromal keratocyte due to the loss of stability factor Human antigen R.
After the removal of epithelium and endothelium, the stroma from a normal cornea was incubated overnight in collagenase. The cells were then seeded in a 6-well plate in DMEM medium containing 10% fetal bovine serum and 1% antibiotics, and were maintained at 37°C in a 5%-CO2-humidified air. The Silencer® Select siRNA-mediated knockdown of β-actin, HuR, GAPDH were performed according to a manufacture protocol. The cells were transiently transfected with 5 nm (final concentration) of either the control (siCTR) or siRNA that specifically targeted HuR mRNA, β-actin mRNA or GAPDH mRNA. The cells were processed after 72 hr post-transfection. For immunohistochemical analysis, the siRNA treatment was done on cover slips and the expression levels of β-actin and γ-actin after knockdown of β-actin and Human antigen R (HuR) were analyzed by using a confocal microscopic imaging method.
Our previous results have shown that the expression of β-actin gene was down-regulated in the stroma of the keratoconus corneas but not in stroma of normal and Fuch’s dystrophic corneas. We also observed that HuR was down-regulated in keratoconus stroma. The siRNA mediated-knockdown of HuR resulted in a down-regulation of β-actin as seen in the confocal images. However, γ-actin level was unaffected on the siRNA-mediated knockdown of either β-actin or HuR. GAPDH and siCTR was used as a positive and negative control, respectively for the transfection and confocal image analysis.
β-actin mRNA has a long half life and HuR binding to U-rich element is involved in its mRNA stability. The above result of knockdown experiments show for the first time that β-actin down-regulation in human keratocytes is mediated through HuR.
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