Transfection of Short Interfering RNA to Supress ICAM–1 in Human Retinal Endothelial Cells

Abstract

Abstract: : Purpose: Short interfering RNAs (siRNAs) are powerful sequence specific reagents that suppress gene expression in mammalian cells. The purpose of this study is to demonstrate that transfection of siRNAs into human retinal endotherial cells (HRECs) specifically reduced endogeneous expression of ICAM–1 upon cytokine stimulation. Methods:HRECs were cultured and seeded in the plates, the next day transfection was performed with ICAM–1 specific siRNA and Lipofectamine 2000. IL–1ß was added to the medium for stimulation of ICAM–1. After 24 hours, HRECs were collected and determined ICAM–1–specific target protein by ELISA. Total RNA was extracted from another HRECs and ICAM–1 specific target RNA was determined by quantitative RT–PCR. Fluorescein–conjugate control siRNA combined with lipofectamine 2000 was transfected in HRECs, after 24 hours, HRECs were observed by fluorescein microscopy. Results: Protein expression of ICAM–1 was significantly reduced in transfected ICAM–1 siRNA cells compared with control cells. Messenger–RNA expression was also reduced up to 50% by ICAM–1 siRNA. Fluorescein–conjugate control siRNA was transfected into the HRECs especially into the nucleus. Conclusions: Short interfering RNA suppressed gene expression of ICAM–1 in HRECs, this finding suggests that RNA–targeting approach may provide a novel therapeutic option for reinal neovascularization and ischemic retinal disorders.

Keywords: vascular cells • cell adhesions/cell junctions • retina