RNA Interference of Transforming Growth Factor–β2 in Human Respiratory Fibroblasts

Abstract

Transforming growth factor-β2 (TGF-β2) is a principal cytokine of interest in the pathogenesis of scar formation and is a potential target for future molecular-based adjunctive therapies. The authors hypothesize that interfering RNA (RNAi) can be used to modulate TGF-β2 production in cultured human respiratory fibroblasts. Basic science. Setting. Laboratory. RNAi constructs targeted to the TGF-β2 transcript were complexed with microsomal lipids and applied to human fibroblasts in cell culture. Transfection efficiency and cell viability were measured by fluorescence microscopy. Messenger RNA (mRNA) for TGF-β2 was measured 48 hours posttransfection using real-time quantitative PCR. The quantity of TGF-β2 protein produced with increasing concentrations of RNAi was measured using enzyme-linked immunosorbent assay. The function of RNAi-treated fibroblasts was tested using a wound-healing assay. Transfection efficiency of more than 80% was achieved with minimal induced cell death. Treated cells showed selective knockdown of 80% of TGF-β2 mRNA, which was confirmed with negative controls. As the concentration of RNAi was increased, an incremental decrease in TGF-β2 protein was measured. RNAi technology is an effective means of localized and transient gene silencing in cultured human fibroblasts. Transfection can be achieved using microsome complexed RNAi with minimal induced cell death. This preliminary result shows promise for future in vitro studies.