Effect of small interfering RNA on the expression of connective tissue growth factor and type I and III collagen in skin fibroblasts of patients with systemic sclerosis

Abstract

Background: Systemic sclerosis (SSc) is characterized by an excessive production of extracellular matrix. It is widely accepted that fibrosis is induced by transforming growth factor (TGF)-beta in the early stage and is subsequently maintained by connective tissue growth factor (CTGF). CTGF is a cysteine-rich mitogenic peptide that has been involved in various fibrotic disorders and can be induced in fibroblasts by activation with TGF-beta.

Objectives: To evaluate the effect of small interfering RNA (siRNA) targeting CTGF on the expression of CTGF and type I and type III collagen in SSc.

Methods: Skin fibroblasts from patients with SSc were cultured in vitro and later transfected using four CTGF-specific siRNAs and one nonspecific siRNA. The effect of CTGF-specific siRNAs on the expression of CTGF and type I and type III collagen was examined and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry.

Results: Semiquantitative RT-PCR analysis showed that the four CTGF-specific siRNAs significantly reduced CTGF mRNA expression (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 73%. Three of the four siRNAs could also depress the transcriptional levels of type I and type III collagen mRNA (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 37% and 29% for type I and type III collagen, respectively. Western blot analysis further demonstrated that three CTGF-specific siRNAs could significantly decrease CTGF protein level (P < 0.001). In addition, immunocytochemical analysis showed that the expression of type I collagen was significantly decreased in fibroblasts after transfection with siRNA742, whereas inhibition of expression of type III collagen was modest.

Conclusions: Our data for the first time showed that CTGF RNA interference could inhibit expression of CTGF and type I and III collagen in SSc fibroblasts and indicated that CTGF might be an upstream factor regulating type I and type III collagen synthesis, particularly type I collagen. Our findings suggest that silencing CTGF expression might facilitate a potential therapeutic approach for SSc.