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Roy Joseph, Om Srivastava, Roswell Pfister, ; Up-Regulation of Apoptotic Markers Following β-Actin Gene-Silencing in Corneal Stromal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5242.
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To determine whether apoptotic markers are up-regulated in normal corneal stromal fibroblasts after β-actin gene silencing.
Normal human primary corneal stromal fibroblasts were generated. The fibroblasts 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 knock-down of β-actin and siCTR (si-control, negative control) 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 β-actin mRNA. The cells were processed after 72 h post-transfection and the RNA extracted reverse transcribed to cDNA for quantitative real-time PCR. The mRNA expression levels of Caspase-9 and Caspase-3 were measured. The apoptotic cell death was analyzed using Tunel assay after the above described siRNA treatments.
Our previous results had shown that siRNA mediated-knock down of β-actin gene resulted in a significant decrease in cell proliferation and their migration. RT-PCR and Quantitative RT-PCR analyses showed a 2-fold increase in the expression of mRNA of caspase-3 at 96 h, and also of caspase-9 after β-actin gene-silencing. An increase in Tunel positive cells compared to siCTR was also observed.
Our previous results had shown that the expression of β-actin gene was down-regulated in the stroma of the keratoconus corneas but not in stroma of normal corneas (Invest. Ophthalmol. Vis. Sci. 2012, 53; 4032-4041). The above results show that β-actin gene-silencing results in an increased apoptosis of corneal fibroblasts, which seems to be a caspase-3- mediated mechanism.
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