Abstract
Purpose :
Complement factor H (CFH) has been associated with increased risk for age related macular degeneration (AMD). CD46, a complement regulatory protein, is located at the same sub-band on the same chromosome as CFH and they lie within a complex of immunoregulatory genes. The purpose of this study is to elucidate the biological function of CFH and CD46 in RPE through CFH or CD46 knockdown and microarray studies.
Methods :
Human ARPE19 cells were cultured to 70% confluence. CFH and CD46 siRNAs were used to knock down CFH or CD46 in ARPE19. After 48 hours of CFH siRNA transfection, ARPE19 were collected and total RNA were isolated, cDNA and Biotin-labeled antisense cRNA produced, target hybridization, washing, staining and scanning probe arrays were done using standard technique. Affymetrix GCOS Manager and other online bioinformatic software system were used for data analysis.
Results :
After 48 hours of siRNA transfection, CFH or CD46 protein expression in ARPE-19 cells was decreased to more than 70% of the control levels. The analyses of gene expression profiles between CHF- and random- siRNA knockdown ARPE-19 point to alterations of genes such as cyclin D1 (CCND1), cadherin, frizzled class receptor 7 (FZD7), glutathione S-transferase alpha 5 (GSTA5), interactor of little elongation complex ELL subunit 2 (ICE2), integrin beta 4 (ITGB4), myelin basic protein (MBP), protein kinase, cAMP-dependent catalytic alpha (PRKACA), proteasome subunit alpha 5 (PSMA5), SR-related CTD-associated factor 11 (SCAF11), tyrosinase-related protein 1 (TYRP1) in cellular pathways for apoptosis, autophagy, cell adhesion, Krebs-TCA cycle, melanin synthesis, proteasome degradation, Wnt signaling. Comparison of gene expression profiles from CD46 and CHF siRNA knockdown in ARPE-19 cells reveal many common and unique pathways are involved.
Conclusions :
The results suggest that CHF and CD46 may play important roles in regulating numerous vital cellular activities to maintain the normal function in pigment epithelium. Alterations in CHF or CD46 expression may lead to secondary changes in other genes of the RPE, and may contribute to the pathogenesis of diseases such as age related macular degeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.