간성상세포의 활성에서 siRNA를 이용한 TGF-β1의 전사조절
Transcriptional Regulation of Hepatic Stellate Cell Activation by siRNA for TGF-β1
1
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Background : The cytokine-induced activation of hepatic stellate cells (HSC) plays a major role in liver fibrosis. Quiescent HSCs undergo phenotypic transformation called “transdifferentiation” in response to viral, chemical or immune insults to the liver. The cytokine TGF-β1 plays a key role in progressive liver fibrosis. Since small interfering RNA (siRNA) is a powerful tool for silencing gene expression post-transcriptionally, the present study aimed to determine whether synthetic TGF-β1 siRNA down-regulates the expression of the TGF-β1 gene in immortalized and activated rat HSCs (HSC-T6s). The study examined whether synthetic TGFβ1 siRNA prevents rat HSCs activation and extracellular matrix (ECM) production. Methods : TGF-β1 siRNA or a control (pU6) siRNA was added to HSC-T6 culture media. We then performed RT-PCR and western blot analyses for TGF-β1 and ECM components (fibronectin, type-I collagen, and TIMP-1). Results : TGF-β1 siRNA significantly down-regulated expression of TGF-β1 mRNA and protein and attenuated mRNA and protein expressions of type-I collagen, fibronectin, and TIMP-1, as compared to the control. Conclusions : TGF-β1 siRNA can effectively down-regulate the expression of TGF-β1 in rat HSC, resulting in significant inhibition of HSC activation and of ECM production. These data indicate that synthetic TGF-β1 siRNA can be a useful treatment modality to prevent liver fibrosis.
Abstract
Background : The cytokine-induced activation of hepatic stellate cells (HSC) plays a major role in liver fibrosis. Quiescent HSCs undergo phenotypic transformation called “transdifferentiation” in response to viral, chemical or immune insults to the liver. The cytokine TGF-β1 plays a key role in progressive liver fibrosis. Since small interfering RNA (siRNA) is a powerful tool for silencing gene expression post-transcriptionally, the present study aimed to determine whether synthetic TGF-β1 siRNA down-regulates the expression of the TGF-β1 gene in immortalized and activated rat HSCs (HSC-T6s). The study examined whether synthetic TGFβ1 siRNA prevents rat HSCs activation and extracellular matrix (ECM) production. Methods : TGF-β1 siRNA or a control (pU6) siRNA was added to HSC-T6 culture media. We then performed RT-PCR and western blot analyses for TGF-β1 and ECM components (fibronectin, type-I collagen, and TIMP-1). Results : TGF-β1 siRNA significantly down-regulated expression of TGF-β1 mRNA and protein and attenuated mRNA and protein expressions of type-I collagen, fibronectin, and TIMP-1, as compared to the control. Conclusions : TGF-β1 siRNA can effectively down-regulate the expression of TGF-β1 in rat HSC, resulting in significant inhibition of HSC activation and of ECM production. These data indicate that synthetic TGF-β1 siRNA can be a useful treatment modality to prevent liver fibrosis.
참고문헌 (30)
- Friedman SL, "Liver fibrosis-from bench to bedside", J Hepatol, 38(1), pp.S38~S53 (2003)
- Reeves HL, "Activation of hepatic stellate cells-a key issue in liver fibrosis", Front Biosci, 7, pp.d808~d826 (2002)
- Gressner AM, "Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-b as major players and therapeutic targets", J Cell Biol Med, 10, pp.76~99 (2006)
- Vogel S, "An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro", J Lipid Res, 41, pp.882~893 (2000)
- Aterman K, "The parasinusoidal cells of the liver: a historical account", Histochem J, 18, pp.279~305 (1986)
- Bataller R, "Liver fibrosis", J Clin Invest, 115, pp.209~218 (2005)
- Gressner AM, "Perisinusoidal lipocytes and fibrogenesis", Gut, 35, pp.1331~1333 (1994)
- Sanderson N, "Hepatic expression of mature transforming growth factor b1 in transgenic mice results in multiple tissue lesions", Proc Natl Acad Sci, pp.2572~2576 (1995)
- George J, "In vivo inhibition of rat stellate cell activation by soluble transforming growth factor b type II receptor: a potential new therapy for hepatic fibrosis", Proc Natl Acad Sci, pp.12719~12724 (1999)
- Qi Z, "Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat", Proc Natl Acad Sci, pp.2345~2349 (1999)
- Nakamura T, "Inhibition of transforming growth factor b prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats", Hepatology, 32, pp.247~255 (2000)
- Arias M, "Adenoviral expression of a transforming growth factor-b1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats", BMC Gastroenterology, 3, pp.29~ (2003)
- Dave RS, "RNA interference: on the road to an alternate therapeutic strategy!", Rev Med Virol, 13, pp.3~85 (2003)
- Elbashir SM, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells", Nature, 411, pp.494~498 (2001)
- Shackel NA, "Intrahepatic gene silencing by RNA interference", Gastroenterology, 126, pp.356~358 (2004)
- Lewis DL, "Efficient delivery of siRNA for inhibition of gene expression in postnatal mice", Nat Genet, 32, pp.107~108 (2002)
- Song E, "RNA interference targeting Fas protects mice from fulminant hepatitis", Nat Med, 9, pp.347~351 (2003)
- Zender L, "Caspase 8 small interfering RNA prevents acute liver failure in mice", Proc Natl Acad Sci, pp.7797~7802 (2003)
- Kim KH, "The antifibrotic effect of TGFb1 siRNAs in murine model of liver cirrhosis", Biochem Biophys Res Commun, 343, pp.1072~1078 (2006)
- Yun HS, "Cytotoxic effects of the conjugated linoleic acid isomers t10c12, c9t11-CLA and mixed form on rat hepatic stellate cells and CCl4-induced hepatic fibrosis", J Nutri Biochem, 19, pp.175~183 (2008)
- Paddison PJ, "Stable suppression of gene expression by RNAi in mammalian cells", Proc Natl Acad Sci, pp.1443~1448 (2002)
- Brummelkamp TR, "A system for stable expression of short interfering RNAs in mammalian cells", Science, 296, pp.550~553 (2002)
- Paul CP, "Effective expression of small interfering RNA in human cells", Nat Biotechnol, 20, pp.505~508 (2002)
- Jeong KS, "Therapeutic target for chronic liver fibrosis by regulation of transforming growth factor-beta", Basic Appl Pathol, 1, pp.56~60 (2008)
- Bissell DM, "Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation", J Clin Invest, 96, pp.447~455 (1995)
- Bissell DM, "Transforming growth factor beta and the liver", Hepatology, 34, pp.859~867 (2001)
- Gao C, "Transforming growth factor beta (TGF-beta) expression in isolated and cultured rat hepatocytes", J Cell Physiol, 167, pp.394~405 (1996)
- Friedman SL, "Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury", J Biol Chem, 275, pp.2247~2250 (2000)
- Dooley S, "Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2", 3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGF beta signal transduction during transdifferentiation of hepatic stellate cells, 2001, pp.502, 4~10
- Liu C, "Smads 2 and 3 are differentially activated by transforming growth factor-b (TGF-b) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-bindependent", J Biol Chem, 278, pp.11721~11728 (2003)