722. Silencing of Transforming Growth Factor-β Receptor II by RNA Interference Suppresses Extracellular Matrix Production in Rat Hepatic Stellate Cells
Authors: Jin Wook Kim, Song-Qing He, Mark A. Zern, John J. Rossi, Jian Wu
Journal: Molecular Therapy (2006)

BACKGROUND: Transforming growth factor-β (TGF-β) is a major pro-fibrogenic cytokine that mediates the activation of hepatic stellate cells (HSC) and their over-production of extracellular matrix (ECM) components, the two key events during hepatic fibrogenesis. Signal transduction of TGF-β is initiated by binding of the ligand to TGF-β type II receptor (TGFβRII). The aim of this study is to reduce ECM production in rat HSC by knocking-down TGFβRII with RNA interference (RNAi) strategies. METHODS: Selected siRNA were transfected in a rat HSC line, SBC10, using TransIT TKO transfecting reagent. Real-time RT-PCR was used to determine mRNA levels of TGFβRII, procollagen type I (α1) (Procoll-I) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in cells after siRNA or shRNA transfection. shRNA expression cassettes and lentiviral TGFβRII shRNA vectors containing the most effective siRNA sequences were generated to test their effects in SBC-10 cells. RESULTS: TGFβRII gene expression was reduced to 10.7 ± 4.5 % of controls with siRNAs at 50 nM 48 hrs after transfection. Moreover, mRNA levels of Procoll-I and TIMP-1 were reduced to 52.1 ± 6.6 % and 32.8 ± 5.2 % of controls 48 hrs after the siRNA transfection. Transfection of an shRNA expression cassette in SBC10 cells suppressed mRNA levels of TGFβRII, Procoll-I and TIMP-1 by 79%, 23% and 20%, 48 hours after transfection. The lentiviral TGFβRII shRNA vector transduction led to a 91-fold increase in shRNA production 5 days after transduction as indicated by real time RT-PCR, which resulted in a 69% reduction in TGFβRII mRNA levels, and 25–35% reduction in Procoll-I and TIMP-1 mRNA levels in the transduced cells. GFP expression in the lentiviral TGFβRII shRNA vector-transduced cells lasted for 21 days (the length of the experiment), with a transduction rate of 94.3%, as determined by flow cytometry. CONCLUSIONS: The findings demonstrated that the TGFβRII gene silencing by RNAi strategies inhibits fibrogenesis in rat HSCs. This novel approach may offer a new molecular therapy for hepatic fibrosis, for which effective treatment is not readily available. BACKGROUND: Transforming growth factor-β (TGF-β) is a major pro-fibrogenic cytokine that mediates the activation of hepatic stellate cells (HSC) and their over-production of extracellular matrix (ECM) components, the two key events during hepatic fibrogenesis. Signal transduction of TGF-β is initiated by binding of the ligand to TGF-β type II receptor (TGFβRII). The aim of this study is to reduce ECM production in rat HSC by knocking-down TGFβRII with RNA interference (RNAi) strategies. METHODS: Selected siRNA were transfected in a rat HSC line, SBC10, using TransIT TKO transfecting reagent. Real-time RT-PCR was used to determine mRNA levels of TGFβRII, procollagen type I (α1) (Procoll-I) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in cells after siRNA or shRNA transfection. shRNA expression cassettes and lentiviral TGFβRII shRNA vectors containing the most effective siRNA sequences were generated to test their effects in SBC-10 cells. RESULTS: TGFβRII gene expression was reduced to 10.7 ± 4.5 % of controls with siRNAs at 50 nM 48 hrs after transfection. Moreover, mRNA levels of Procoll-I and TIMP-1 were reduced to 52.1 ± 6.6 % and 32.8 ± 5.2 % of controls 48 hrs after the siRNA transfection. Transfection of an shRNA expression cassette in SBC10 cells suppressed mRNA levels of TGFβRII, Procoll-I and TIMP-1 by 79%, 23% and 20%, 48 hours after transfection. The lentiviral TGFβRII shRNA vector transduction led to a 91-fold increase in shRNA production 5 days after transduction as indicated by real time RT-PCR, which resulted in a 69% reduction in TGFβRII mRNA levels, and 25–35% reduction in Procoll-I and TIMP-1 mRNA levels in the transduced cells. GFP expression in the lentiviral TGFβRII shRNA vector-transduced cells lasted for 21 days (the length of the experiment), with a transduction rate of 94.3%, as determined by flow cytometry. CONCLUSIONS: The findings demonstrated that the TGFβRII gene silencing by RNAi strategies inhibits fibrogenesis in rat HSCs. This novel approach may offer a new molecular therapy for hepatic fibrosis, for which effective treatment is not readily available.