RNA interfering connective tissue growth factor prevents rat hepatic stellate cell activation and extracellular matrix production

Abstract

Connective tissue growth factor (CTGF) is a possible key determinant of progressive fibrosis. Small interfering RNA (siRNA) is a powerful tool for silencing gene expression post-transcriptionally. Therefore, the present study aimed to determine whether synthetic siRNA target CTGF down-regulates the expression of the CTGF gene in primary rat hepatic stellate cells (HSC) and HSC T6, and, furthermore, whether it prevents rat HSC activation and extracellular matrix (ECM) production. Primary HSC were obtained by enzymatic perfusion of rat liver. HSC T6, primary HSC were treated with siRNAs that target CTGF or a control siRNA by addition to the culture medium. We obtained one siRNA that could sequence-specifically reduce target gene expression by over 90% at a concentration of 200 nM in the cell culture medium for a total of three siRNAs targeting CTGF genes. In HSC T6 cells, the effect of CTGF siRNA was dose-dependent (50-200 nM) and time-limited to a 24-72-h period. The siRNA knockdown of CTGF significantly reduced the expression of alpha-smooth muscle actin protein, increased the number of cells, upregulated the ratios of G0/G1 stage in rat HSC at 7 days of culture after plating, and attenuated the expression of type I and III collagen mRNA with a supernatant concentration of hyaluronic acid, and type III procollagen in an activated HSC of culture for 24-72 h. CTGF siRNA could effectively and sequence-specifically down-regulate the expression of CTGF in rat HSC, resulting in significant inhibition of HSC activation and proliferation as well as ECM production. These findings indicate that synthetic siRNA targeting CTGF could prove to be a useful treatment of liver fibrosis.