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L. V. Del Priore, H. Cai; CD46 (MCP) Knockdown Alters Genes Involving Cell Localization, Immune System Processing and Biological Adhesion in Human Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2010;51(13):6191.
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© ARVO (1962-2015); The Authors (2016-present)
The complement system has been implicated in the pathogenesis of age-related macular degeneration (AMD). CD46 is a complement regulatory protein located on the basal surface of the retinal pigment epithelium, and thus occupies an important anatomic position between the pigment epithelium and the choriocapillaris external to the outer blood-retinal barrier. The purpose of this study is to elucidate the biological function of CD46 in RPE through CD46 knockdown and microarray studies.
Human ARPE19 cells were cultured to 70% confluence. CD46 siRNAs were designed and synthesized from commercial sources and used to knock down CD46 in ARPE19. Amine was used to transfect the siRNA oligos into cultured ARPE19. After 48 hours of CD46 siRNA transfection, ARPE19 were collected and total RNA were isolated using standard technique. First and second strand cDNA were synthesized with a T7-(dT)24 oligomer for priming. Biotin-labeled antisense cRNA was produced by in vitro transcription. Target hybridization, washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Affymetrix GCOS Manager and Genesifter software system were used for data analysis.
After 48 hours of siRNA transfection, CD46 protein expression in ARPE19 was decreased to 30% of the control level. The analyses of gene expression profiles between CD46- and random siRNA-knockdown ARPE19 point to alterations of genes such as centrosomal protein (CEP57), SECIS binding protein 2 (SECISBP2), interleukin 6 signal transducer (IL6ST), chitotriosidase (CHIT1), ARP2 actin-related protein homolog (ACTR2), neurofibromin (NF1) and cyclin-dependent kinase (CDK6) in cellular pathways for regulating cell localization, immune responses and biological adhesion.
The results suggest that CD46 on RPE cells may play an important role in regulating numerous vital cellular activities to maintain the normal function of the pigment epithelium. Alterations in CD46 expression may lead to secondary changes in other genes of the RPE, and may thus contribute to the pathogenesis of diseases such as age related macular degeneration.
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