Ningning Yang1, Ram I Mahato. 1. Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 South Manassas, Memphis, Tennessee 38103-3308, USA.
Abstract
PURPOSE: The objective was to determine the role of promoters and miRNA backbone in shRNA-based hepatic stellate cell (HSC)-specific transforming growth factor (TGF)-β1 gene silencing. This is expected to avoid the side effect of non-specific TGF-β1 gene silencing. METHODS: Two most potent shRNAs targeting 769 and 1033 start sites of rat TGF-β1 mRNA were cloned into pSilencer 1.0 vector for enhanced TGF-β1 gene silencing. We then constructed HSC-specific pri-miRNA mimic and pri-miRNA cluster mimic expression plasmids in which shRNA expression was driven by a glial fibrillary acidic protein (GFAP) promoter to achieve HSC-specific TGF-β1 gene silencing to avoid nonspecific inhibition of TGF-β1 expression in other cells and organs. RESULTS: These TGF-β1 pri-miRNA-producing plasmids showed the inhibition of proliferation and induced apoptosis of activated HSC-T6 cells. TGF-β1 pri-miRNA cluster mimic plasmids decreased TGF-β1 and collagen gene expression at both mRNA and protein levels. CONCLUSIONS: GFAP promoter driven TGF-β1 pri-miRNA producing plasmids have the potential to be used for site-specific gene therapeutics to treat liver fibrosis.
PURPOSE: The objective was to determine the role of promoters and miRNA backbone in shRNA-based hepatic stellate cell (HSC)-specific transforming growth factor (TGF)-β1 gene silencing. This is expected to avoid the side effect of non-specific TGF-β1 gene silencing. METHODS: Two most potent shRNAs targeting 769 and 1033 start sites of rat TGF-β1 mRNA were cloned into pSilencer 1.0 vector for enhanced TGF-β1 gene silencing. We then constructed HSC-specific pri-miRNA mimic and pri-miRNA cluster mimic expression plasmids in which shRNA expression was driven by a glial fibrillary acidic protein (GFAP) promoter to achieve HSC-specific TGF-β1 gene silencing to avoid nonspecific inhibition of TGF-β1 expression in other cells and organs. RESULTS: These TGF-β1 pri-miRNA-producing plasmids showed the inhibition of proliferation and induced apoptosis of activated HSC-T6 cells. TGF-β1 pri-miRNA cluster mimic plasmids decreased TGF-β1 and collagen gene expression at both mRNA and protein levels. CONCLUSIONS:GFAP promoter driven TGF-β1 pri-miRNA producing plasmids have the potential to be used for site-specific gene therapeutics to treat liver fibrosis.
Authors: Anne-Charlotte de Gouville; Valerie Boullay; Gael Krysa; Julia Pilot; Jean-Marie Brusq; Florence Loriolle; Jean-Michel Gauthier; Stephen A Papworth; Alain Laroze; Françoise Gellibert; Stephane Huet Journal: Br J Pharmacol Date: 2005-05 Impact factor: 8.739
Authors: Francesco P Russo; Malcolm R Alison; Brian W Bigger; Eunice Amofah; Aikaterini Florou; Farhana Amin; George Bou-Gharios; Rosemary Jeffery; John P Iredale; Stuart J Forbes Journal: Gastroenterology Date: 2006-05 Impact factor: 22.682