Abstract
Abstract: :
Purpose: FABP5 is an intracellular lipid transport protein whose functions have been associated with fatty acid signaling, cell growth, and regulation. Since we found upregulation of FABP5 in our D–galactose model of aging, we hypothesized that FABP5 is involved in RPE/choroid aging. The purpose of this study was to demonstrate the feasibility of RNA interference mediated by FABP5 siRNA as a potential therapy in a human retinal pigment epithelium (RPE) cell line (ARPE_19). Methods: The distribution of FABP5 in the ARPE–19 cells and histopathologic samples were confirmed by Western blot and immunochemical staining, respectively. The expression of FABP5 gene was detected by RT–qPCR using RNA isolated from laser captured human RPE cells. ARPE–19 cells were transfected with the pooled FABP5 siRNA that consisted of four individual siRNAs I, II, III, and IV. The transfection efficacy in ARPE–19 cells was visualized using a Fluorescent oligo. The toxicity of siRNA was measured by a Hoechst dye that labels dying cells. Dose response experiments were performed from 0.001 to 100 nM of the pooled siRNA. The inhibitory effect and duration of the 10nM pooled and individual (I, II, III, IV) siRNAs were observed during 24–96h following a 24h–siRNA treatment. The purity of RNA was estimated by the 260/280 ratio. The relative FABP5 mRNA expression in comparison with a scrambled negative control siRNA was determined by RT–qPCR, which was normalized to GAPDH in the samples. The protein level of FABP5 was quantified by flow cytometry. Results: Immunohistochemical staining of human fundus sections showed strong labeling of FABP5 within the RPE and choriocapillaris endothelium. The mRNA of FABP5 was expressed in situ. In ARPE–19 cells, the transfection efficacy was > 80%, as indicated by intracellular fluorescent labeling. The cell death rate after a 24h–siRNA treatment was < 2%, and was equal to siRNA negative controls. The pooled siRNA.FABP5 effectively inhibited the FABP5 mRNA and protein expression after a 24h–siRNA treatment. The inhibitory rate on mRNA was > 80 %, which was maintained for 96h following a 24h–siRNA treatment; the amount of FABP5 protein was decreased about 50% after 24h. The inhibitory rates on mRNA of siRNAs I, II, III, and IV were 73%, 78%, 80%, and 73%, respectively. Conclusions: This work suggests that siRNA can specifically target FABP5 mRNA to inhibit the expression of the RNA and protein of FABP5 in a human RPE cell line.
Keywords: retinal pigment epithelium • metabolism • gene/expression