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TGF-beta1 gene silencing for treating liver fibrosis.

ABSTRACT: Small interfering RNA (siRNA) and short hairpin RNA (shRNA) targeting different regions of transforming growth factor beta1 (TGF-beta1) mRNA were designed and the silencing effect was determined after transfection into immortalized rat liver stellate cells (HSC-T6). There was not only significant decrease in TGF-beta1, tissue inhibitor of metalloproteinase 1 (TIMP-1), alpha-smooth muscle actin (alpha-SMA) and type I collagen after transfection with TGF-beta1 siRNAs, but also synergism in gene silencing when siRNAs targeting two different start sites were used as a pool for transfection. The two siRNA sequences which efficiently inhibited TGF-beta1 gene expression were converted to shRNAs via cloning into the pSilencer1.0. There was significant decrease in TGF-beta1 and TIMP-1 when HSC-T6 cells were transfected with pshRNA targeting the same regions of TGF-beta1 mRNA as siRNAs. Furthermore, TGF-beta1 gene silencing in HSC-T6 cells significantly decreased the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). In conclusion, both siRNA and shRNA showed sequence-specific and dose dependent TGF-beta1 gene silencing and have the potential to treat liver fibrosis.

SUBMITTER: Cheng K

PROVIDER: S-EPMC2743970 | biostudies-literature | 2009 May-Jun

REPOSITORIES: biostudies-literature

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TGF-beta1 gene silencing for treating liver fibrosis.

Cheng Kun K Yang Ningning N Mahato Ram I RI

Molecular pharmaceutics 20090501 3

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) targeting different regions of transforming growth factor beta1 (TGF-beta1) mRNA were designed and the silencing effect was determined after transfection into immortalized rat liver stellate cells (HSC-T6). There was not only significant decrease in TGF-beta1, tissue inhibitor of metalloproteinase 1 (TIMP-1), alpha-smooth muscle actin (alpha-SMA) and type I collagen after transfection with TGF-beta1 siRNAs, but also synergism in gene si ...[more]

PMID: 19388665

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Project description:BackgroundLiver fibrosis represents a significant and severe health care problem and there are no efficient drugs for therapy so far. Preventing the progression of fibrogenesis and revival endogenous repair activities is an important strategy for both current and future therapies. Many studies of liver fibrosis consist of animal testing with various hepatotoxins. Although this method is often used, the model at which cirrhosis or extensive fibrosis becomes irreversible has not been well defined and is not representative of early-stage fibrogenesis. We here report the establishment of a transient and reversible liver fibrosis animal model which may better represent an early and natural fibrotic event. We used a high-speed intravenous injection of naked plasmid DNA of transforming growth factor-beta1 (TGF-beta1) gene which is under the control of a metallothionein-regulated gene in a pPK9A expression vector into the tail vein (the hydrodynamics-based transfer) and fed the mouse with zinc sulfate (ZnSO4)-containing water simultaneously.ResultsUsing our hydrodynamics-based gene transfer model we found that upon induction by ZnSO4, the serum TGF-beta1 level in Balb/c mice and Sp1 transcription factor binding activity peaked at 48 h and declined thereafter to a normal level on the 5th day. In addition, mRNA and protein levels of TGF-beta1 in the liver were also upregulated at 48 h. Furthermore, induction of TGF-beta1 increased the alpha-smooth muscle actin (alpha-SMA), p-Smad2/3, hydroxyproline and collagen 1A2 (Col 1A2) levels in the liver, suggesting a significant liver fibrosis.ConclusionOur results show that TGF-beta1 in pPK9a-transferred mice liver with ZnSO4 feeding can achieve a high expression level with significant fibrosis. However, since TGF-beta1 induction is transient in our model, the fibrotic level does not reach a large scale (panlobular fibrosis) as seen in the CCl4-treated liver. Our model hence represents a dynamic and reversible liver fibrosis and could be a useful tool for studying early molecular mechanism of fibrogenesis or screening of antifibrotic drugs for clinical use.

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TGF-beta1 gene silencing for treating liver fibrosis.

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TGF-beta1 gene silencing for treating liver fibrosis.

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