Evaluation of therapeutic effect of PCBP2 siRNA on hepatic stellate cells and development of a streptavidin-based siRNA delivery system
No Thumbnail Available
Authors
Meeting name
Sponsors
Date
Journal Title
Format
Thesis
Subject
Abstract
Type I collagen accumulates during liver fibrosis primarily because α-complex protein-2 (αCP2), encoded by the poly(rC) binding protein 2 (PCBP2) gene, binds to the 3′end of the collagen mRNA and increases its half-life. Study in chapter 3 aimed to reverse the pro-fibrogenic effect of alcohol on hepatic stellate cells (HSCs) by silencing the PCBP2 gene with siRNA. The silencing effects of a series of predesigned PCBP2 siRNAs were evaluated in the rat hepatic stellate cell line, HSC-T6. The pro-fibrogenic effects of alcohol on the expression levels of PCBP2 and type-I collagen were examined by several methods. The effect of PCBP2 siRNA on the stability of type I collagen α1(I) mRNA was investigated by an in vitro mRNA decay assay. We identified one potent PCBP2 siRNA that reversed the alcohol-induced expression of PCBP2 in HSCs. The decay rate of the collagen α1(I) mRNA increased significantly in HSCs treated with the PCBP2 siRNA. This study provides the first evidence that alcohol up-regulates the expression of PCBP2, which subsequently increases the half-life of collagen α1(I) mRNA. Silencing of PCBP2 using siRNA may provide a promising strategy to reverse the alcohol-induced pro-fibrogenic effects in HSCs. Furthermore, we developed a streptavidin-based nanocomplex that can efficiently deliver the PCBP2 siRNA to HSCs. Biotin−siRNA and biotin−cholesterol were mixed with streptavidin to form the streptavidin−biotin complex, which was further condensed electrostatically with positively charged protamine to form the final multicomponent siRNA nanocomplex in the size range of 150−250 nm. The siRNA nanocomplex does not induce cytotoxicity in rat HSCs as compared to commercially available transfection agents. The cellular uptake efficiency of the siRNA nanocomplex is higher in rat HSCs than other cell lines, such as Caco-2 and PC-3, indicating that receptor-mediated endocytosis mainly contributes to the cellular uptake of the siRNA nanocomplex. The siRNA nanocomplex exhibits more than 85% silencing effect on the PCBP2 mRNA in HSCs. Stability study indicates that the nanocomplex can efficiently protect siRNA from degradation in the serum. The streptavidin-based multicomponent siRNA nanocomplex provides a promising strategy to deliver the PCBP2 siRNA to HSCs. Moreover, the nanocomplex can be used as a platform for other diseases by changing the siRNA sequence and targeting ligand. Understanding of the subcellular distribution of a small interfering RNA (siRNA) formulation is essential for improving the silencing activity of the siRNA. Previously, we have developed a multicomponent streptavidin-based nanocomplex to deliver the PCBP2 siRNA to rat hepatic stellate cells (HSCs) to inhibit the expression of αCP2, a critical protein in alcoholic liver fibrogenesis. This study at chapter 5 aims to evaluate the cellular uptake, subcellular trafficking and intracellular fate of the siRNA and other components, such as protamine and streptavidin, of the nanocomplex. Cellular uptake and silencing activity of the siRNA nanocomplex was compared with commercially available Lipofectamine-2000. siRNA nanocomplexes containing fluorescently labeled siRNA, streptavidin and protamine were used for thealuation of subcellular trafficking of each of the components. The results showed that the silencing activity of the siRNA nanocomplex decreases with incubation time, indicating that part of the siRNA is either entrapped in the endosomes or associated with streptavidin. The intracellular trafficking of the siRNA nanocomplex exhibits divergent cellular distribution of siRNA, protamine and streptavidin. These findings can facilitate the rational design of streptavidin based siRNA delivery systems.
Table of Contents
Introduction -- Review of literature -- PCBP2 siRNA reverses the alcohol-induced pro-fibrogenic effect in hepatic stellate cells -- Development of streptavidin-based nanocomplex for siRNA delivery -- Intracellular trafficking and silencing activity of the siRNA loaded streptavidin nanocomplex -- Summary