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Cristina M. Kenney, Marilyn Chwa, Shari R. Atilano, Janelle M. Pavlis, Anthony Nesburn, Nitin Udar; Downregulation of SCARA3, CPSF3 and FOXM1 in Keratoconus Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1112.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC) corneas have increased levels of oxidative damage. We hypothesized that the increase could be due to an imbalance in either the synthesis or elimination (or a combination of both) of the reactive oxygen/nitrogen species (ROS/RNS). In order to evaluate several pathways involved in this process, we studied the expression profile of three key genes: SCARA3 (a major ROS/RNS scavenger that protects cells against UV light), CPSF3 (a pre-mRNA 3-prime-end-processing endonuclease that is closely associated with SCARA3), and FOXM1 (a transcription factor for antioxidant genes) in KC cells in vitro.
Normal (n=6) and KC (n=5) stromal cells were cultured in Minimal Essential Media with 10% fetal bovine serum to establish primary stromal fibroblast cultures. The RNA was isolated and cDNA synthesized. Quantitative PCR was carried out on the target genes (including their isoforms) and data analyzed using relative quantification. Immunohistochemisty was performed on intact Normal and KC human corneas (n=10) using antibodies to SCARA3 and CSPF3.
In KC cells, the SCARA3 full length variant 1 (NM_016240) had a 3.5 fold decline (p=0.0001) and the SCARA3 variant 2 (NM_182826) showed a 4.2 fold decrease (p=0.0001) compared to the Normal cells. The gene expression for total SCARA3 (NM_016240 plus NM_182826) was significantly decreased compared to Normal cells (> -3 fold, p=0.0001). Expression levels for CSPF3 were significantly reduced in KC cells (-1.9 fold, p=0.014). The KC cells had a 5.5 fold decrease of the total FOXM1 (NM_021953, NM_202002, NM_202003), p=0.0001 and a 3.5 fold decline in the FOXM1 isoform variant 1 (NM_202002) (p=0.008). Immunohistochemistry showed SCARA3 and CPSF3 proteins present in stromal cells of ex vivo Normal and KC corneas.
Increased oxidative stress seen in KC cells may be related to the reduced expressions of SCARA3, a critical ROS/RNS scavenger, as well as FOXM1 which plays an essential role in upregulating antioxidant genes. The lower levels of CPSF3, an enzyme necessary for polyadenylation of RNA, could potentially affect the pre-mRNA processing within the cells and might account for the numerous altered genes reportedly found in KC corneas. This combination would make the KC cells more susceptible to oxidative damage.
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