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The antifibrotic effect of TGF-β1 siRNAs in murine model of liver cirrhosis
BBRC Biochemical and Biophysical Research Communications 343 (2006) 1072–1078 www.elsevier.com/locate/ybbrc
The antifibrotic effect of TGF-b1 siRNAs in murine model of liver cirrhosis Kyung-Hyun Kim a, Hyun-Chul Kim b, Mee-Yul Hwang a, Hoon-Kyu Oh a, Tae-Sung Lee c, Young-Chae Chang a, Ho-Jung Song a, Nam-Hee Won d, Kwan-Kyu Park a,* a
Department of Pathology, Catholic University of Daegu, College of Medicine, 3056-6 Daemyung 4-Dong, Nam-Gu, Daegu 705-718, Republic of Korea b Kidney Institute, Keimyung University School of Medicine, Daegu 700-712, Republic of Korea c Department of Obstetrics and Gynecology, Catholic University of Daegu, College of Medicine, Daegu 705-034, Republic of Korea d Department of Pathology, College of Medicine, Korea Universitiy, 125-1 5-ga Anam-dong Sunguk-gu, Seoul, Republic of Korea Received 10 March 2006 Available online 23 March 2006
Abstract Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-b1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-b1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-b1 siRNAs reduced the expression of TGF-b1 in the livers of CCl4 injection compared with those of control group, and the expression of type I collagen and a-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-b1 siRNAs inhibit TGF-b1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human. 2006 Elsevier Inc. All rights reserved. Keywords: TGF-b1; RNA interference; siRNA; Liver cirrhosis
Liver cirrhosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix (ECM) proteins, which is characteristic of most types of chronic liver diseases [1]. Recent investigations have shown that transforming growth factor (TGF)-b1 is one of the most powerful profibrogenic mediators and plays a major role in the development of liver cirrhosis [2,3]. The pathophysiology of ECM formation during liver cirrhosis is multifaceted and complicated. Therefore, there are numerous attempts to prevent liver cirrhosis including antifibrotic strategies. One of these involves targeting mRNA by using antisense oligodeoxynucleotides (ODNs), which are the sequence-specific binding to target mRNA, resulting in the prevention of gene translation. Also, *
Corresponding author. Fax: +82 53 650 4834. E-mail address: [email protected] (K.-K. Park).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.087
double-stranded ODN (decoy ODN) technology, which has consensus sequence binding of the transcription factor, blocks the activity of a specific transcription factor suppressing gene expression at transcription level. Nevertheless, the greatest limitation of these ODNs is that they are easily degraded by nucleases or by readily nonspecific reaction with the control strand. These problems evoke more nonspecific bindings and adverse effects in vivo [4–6]. To circumvent these problems, we applied another gene silencing method, RNA interference (RNAi), for this study. This is the induction of sequence-specific gene silencing by double-stranded RNA (dsRNA). Long dsRNA molecules are processed to small 21–23 nucleotide interfering RNAs by Dicer, an endogenous RNA III enzyme [7]. Although siRNA has been recently used in mammalian systems to define the functional role of individual genes or diseases [8], in vivo study for liver diseases is not as well characterized as the others.
K.-H. Kim et al. / Biochemical and Biophysical Research Communications 343 (2006) 1072–1078
The aim of this study was to see RNAi phenomena for the prevention of fibrosis in the murine liver cirrhotic model by using siRNAs for TGF-b1. To induce TGF-b1 specific gene silencing, we chose 21-nucleotide complementary sequence with TGF-b1 mRNA ligated into pU6 promoter, which is available to replicate without host immune response and cytotoxicity in vivo. Intraperitoneal CCl4 administration was used for the experiment to induce hepatic fibrosis in the mouse. This study was focussed on the gene silencing and antifibrotic effects of TGF-b1 siRNAs in the murine model of liver cirrhosis. Materials and methods Animal model and protocol. Six-week-old C57BL/6 mice were randomly divided into three groups: a normal group treated nothing (normal control, NC), a hepatic damage group with fibrosis (HD), and a group having hepatic damage with fibrosis treated with TGF-b1 siRNA (THD). The HD group and the THD group received intraperitoneal injection of CCl4 (2 ml/kg) dissolved in corn oil (1:3 ratio) three times a week for 8 weeks. SiRNAs were introduced via tail vein 3 weeks after first CCl4 injection. Mice were sacrificed after every 1 week from siRNA injection and were weighed before sacrifice. All mice were kept at 21-25 C under 12-h dark/light cycles. Construction of TGF-b1 siRNAs. To make TGF-b1 specific gene silencing effect, we chose the complementary 21-nucleotide, 5 0 -AACCAAGGAGACGGAATACAG-3 0 , which does not have crossreactivity with any other genes, has a few secondary structures and has 30–50% of the GC contents. We used the pU6-shX which contains promoter regions of the mouse small nuclear RNA U6 to prolong gene silencing [9]. The selected sequence connected with ttcaagaga 9-mer loop and the end of sequence TTTT, which terminates transcription. This structure cloned into EcoRI, XbaI sites of pU6shX. Transfection of TGF-b siRNAs to the animal and its identification. The deliveries of siRNA were performed through tail vein injection. To confirm the transfection site, the plasmid vector labeled with fluorescence using Mirus (MirusBio, WI, USA). After 24 h of injection, we removed liver from mouse, deep-frozen with OCT compound (Sakura Finetechnical Co., Tokyo, Japan), and sectioned 4 lm thickness and then observed with fluorescence microscopy. Biochemical analysis. Blood samples were obtained from the inferior vena cava. Serum was collected from blood sample by immediate centrifugation at 10,000g for 8 min at 4 C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using automated techniques. Northern analysis. Total RNA was extracted from mouse liver according to the TRI-zol Reagent described previously (Invitrogen, CA, USA). Twenty micrograms of total RNA from each sample was denatured at 65 C for 10 min, electrophoresed on 1.2% agarose/formaldehyde gel containing ethidium bromide, and transferred to a Hybond-N+membrane (Amersham, NJ, USA). The membrane was hybridized with [32P]-labeled cDNA probes in a solution 7% SDS, 1 mM EDTA, 1% BSA, and 0.5 M NaPO4 at 65 C overnight. The membrane was then washed 2 · SSC containing 0.1% SDS at room temperature 1 min, twice with 0.2 · SSC containing 0.1% SDS at 65 C for 30 min. Then AGPA film (Agfa-Gevaert, N.V, Belgium) was exposed to the membrane. The probes were radiolabeled by random priming method using Prime-a-gene labeling system (Promega Corp., WI, USA) in the [32P]dCTP. The probes were used for PCR amplification. The amplified products were purified after resolving in 1% agarose gel using QIA gel extraction kit (Qiagen, CA, USA). Protein isolation and Western blotting. Frozen tissues were homogenized in an IPH buffer (50 mM pH 8.0 Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 100 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 M DTT). After incubation for 30 min on ice, the samples were centrifuged at 12,000 rpm, at 4 C for 30 min and the supernatant was transferred to a
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new tube. Total protein was measured with a Bio-Rad Bradford kit (BioRad Laboratories, CA, USA), then 50 lg of total protein was run on a 15% SDS–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA) which was blocked with 5% skim milk in TBS-T (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. The membrane was probed with the primary antibody for 3 h, washed several times with TBS-T, and then incubated with a horseradish peroxidase (HRPO)-conjugated secondary antibody. Finally, the membrane was washed and developed with an ECL detection system (Amersham, NJ, USA). The primary antibodies used in this study were TGF-b1 (Santa Cruz Biotechnology, CA, USA), a-SMA (Sigma, St. Louis, USA), and type I collagen (Abcam, MA, USA). Histopathological investigation. Small pieces of liver from each lobe were kept in 10% formalin solution. Paraffin blocks were prepared. Crosssection taken from the blocks was stained with Masson’s Trichrome. Immunohistochemical stain. Paraffin-embedded sections were deparaffinized with xylene, dehydrated in decreasing concentrations of ethanol, and then treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. Tissue sections were processed in 10 mM citrate buffer (pH 6.0) and heated to 100 C for 10 min for antigen retrieval. Sections then were incubated with antibodies against TGF-b1 (Santa Cruz Biotechnology, CA, USA), a-SMA (Sigma, St. Louis, USA), and type I collagen (Abcam, MA, USA) for 1 h at 37 C. The sections were then incubated in an EnVision system (DAKO, CA, USA) for 30 min at 37 C with washing in PBS before each incubation. DAB (3,3 0 -diaminobenzidine tetrahydrochloride) was used as the color reagent, and hematoxylin was used as a counterstain. Statistical analysis. The data are presented as means standard errors. The statistical difference between means was determined using Student’s t test; p < 0.05 was considered significant.
Results Construction of TGF-b1 siRNA and confirmation of transfection in mouse liver For the experiment, we designed four kinds of TGF-b1 siRNAs containing the consensus sequence without any open end expression and examined the effect of the sequence specific siRNA on TGF-b1. The described sequence interfered with 40% of the TGF-b1 expression (data not shown) compared with the empty vector, so we used mainly this sequence vector for the experiment. Then we examined the distribution and efficacy of TGF-b1 siRNAs in the mouse liver by FITC-conjugated TGF-b1 siRNAs using Mirus liposome intravenously. After 24 h of injection, the mouse liver was removed and sectioned 4 lm thick. The presence of siRNAs within the liver was assessed by immunofluorescence microscopy. Biochemical and physiological changes in the liver As seen in morphological observation, the HD group was severely damaged with CCl4 administration but the THD group was only a little damaged. The body weights in the HD and THD groups were lower than in the normal group. The serum AST and ALT levels were significantly increased in the HD groups compared with those of the normal groups, but were significantly decreased after TGF-b1 siRNA treatment (Table 1). Therefore, TGF-b1 siRNA treatment was effective in reducing the hepatic damage.
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Table 1 Serum level of AST/ALT and body weight
AST ALT Body wt (g)
NC
4W
6W
8W
HD
THD
HD
THD
HD
THD
57.3 ± 16.3 36.7 ± 12.6 32 ± 1.6
334.6 ± 55.1 784.3 ± 48.6 24 ± 0.8
135.7 ± 38.7 79.3 ± 8.5 27.5 ± 0.4
651.9 ± 94.1 1002.5 ± 126.8 21.3 ± 1.9
161 ± 29.5 176.3 ± 35.6 27.8 ± 0.8
610 ± 50.2 883 ± 49.8 23.3 ± 0.5
149 ± 17.3 137.3 ± 24.3 30 ± 1.0
A normal group treated nothing (normal control, NC), a hepatic damage with fibrosis (HD), and a TGF-b1 siRNA treated with HD groups (THD).
The inhibitory effect of TGF-b1 in the liver after TGF-b1 siRNA treatment The administration of CCl4 results in chronic damage to the liver with accumulation of ECM and increased expres-
sion of TGF-b1. The transcription level of TGF-b1 mRNA was up-regulated in the HD group, but declined in the THD group (Fig. 1A). Similarly, the TGF-b1 protein levels were significantly increased in the HD group compared with those of the normal group, but this increase was
Fig. 1. TGF-b1 mRNA and protein expression after TGF-b1 siRNA injection. Northern blot shows the inhibitory effect on TGF-b1 mRNA in cirrhotic liver after the injection of TGF-b1 siRNA (A). Western blot also shows the inhibitory effect on TGF-b1 protein in cirrhotic liver by TGF-b1 siRNAs (B). (HD group, CCl4 administration only; THD group, CCl4 administration with TGF-b1 siRNAs.)
Fig. 2. Western blot shows the inhibition of type I collagen (A) and a-smooth muscle actin protein (B) expression in cirrhotic mouse liver by TGF-b1 siRNA injection. (HD group, CCl4 administration only; THD group, CCl4 administration with TGF-b1 siRNAs.)
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attenuated by TGF-b1 siRNA treatment in the THD group (Fig. 1B). The expression of type I collagen and a-smooth muscle actin expression after TGF-b1 siRNA treatment Chronic damage to the liver also affects expression of the type I collagen, which is major ECM protein. Western blots shown in Fig. 2A, type I collagen expression was significantly increased in the HD group compared with that in normal group, but it was decreased after TGF-b1 siRNA treatment in the THD group. To determine the liver fibrogenesis, we also observed the expression level of a-smooth muscle actin (a-SMA). Its expression was significantly increased in the HD group compared with the normal group. However, its expression in the THD group was significantly reduced when compared with that in the HD group at 6 and 8 weeks (Fig. 2B). The histopathological finding As shown in Fig. 3, total collagen fibers in the liver were observed by trichrome stain. In HD group, the liver was damaged gradually. Periportal fibrotic bands, which are detected by Masson’s Trichrome stain, were gradually increased, but were much decreased in the
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THD group against with the HD group. There were only minimal residual periportal bands in the THD group. Therefore, TGF-b1 siRNA treatment moderates liver fibrogenesis. Immunohistochemical finding of TGF-b1, type I collagen, and a-SMA In the HD group, the TGF-b1 positive cells were increased in number and size. In addition, the immunohistochemical stain intensity was also increased compared with the THD group. There were few TGF-b1 positive cells in the THD group (Fig. 4). Type I collagen was increased in periportal area in the HD group. However, it was decreased after TGF-b1 siRNA treatment (Fig. 5). a-SMA was also positive in portal vein and periportal region in the HD group, but not in the THD group (Fig. 6). There were significant decreases of fibrosis-related proteins after TGF-b1 siRNA treatment. Discussion In vivo gene silencing studies have been reported in animal models of choroidal neovascularization [10], lung ischemia–reperfusion injury [11], and neuropathic pain [12].
Fig. 3. Trichrome stain in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of total collagen fiber expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, total collagen fiber was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
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Fig. 4. Immunohistochemistry for TGF-b1 in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of TGF-b1 expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, TGF-b1 was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
Fig. 5. Immunohistochemistry for type I collagen in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of type I collagen expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, type I collagen was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
K.-H. Kim et al. / Biochemical and Biophysical Research Communications 343 (2006) 1072–1078
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Fig. 6. Immunohistochemistry for a-SMA in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of a-SMA expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, a-SMA was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
Recently, Y Takabatake et al. [13] have reported that TGFb1 siRNAs significantly suppressed TGF-b1 mRNA and protein expression, thereby ameliorating the progression of matrix expansion in experimental glomerulonephritis. To make an animal model of liver cirrhosis in this study, intraperitoneal injection of CCl4 was used for chronic liver damage [14]. The CCl4 injection results in an increased necrosis followed by the development of fibrosis. Many factors involved in this event, especially TGF-b1 is the most potent regulator in liver cirrhosis. It regulates ECM proteins by reducing matrix degradation and stimulating matrix accumulation [3,15]. We therefore applied it as a target factor for this experiment using the RNAi principle. The phenomenon of RNAi was first discovered in Caenorhbditis elegans as a response to double-stranded RNA (dsRNA), which resulted in sequence-specific gene silencing [16]. In contrast to C. elegans, where RNAi effects are stable, long lasting, and passed onto the offspring [3], gene silencing by transfected siRNA duplexes in mammalian cells is transient. This is because mammalian cells lack the RNA-dependent RNA polymerases that amplify siRNAs in C. elegans. To get around this problem, vectorbased systems for the introduction and stable expression of siRNA in target cells have been developed [17]. These vectors contain RNA polymerase III promoters that either express sense and antisense strands from separate promoters (tandem type) or express short hairpin RNAs (shRNAs) that are cleaved by the Dicer to produce siRNA.
Using this technique, RNAi seems to be more suitable for knockdown experiments and interferes less with following functional readouts in comparison with other gene silencing method, such as antisense [14]. To obtain the maximum efficacy when we transfect siRNAs to our animal model, we constructed TGF-b1 siRNAs containing RNA III-dependent RNA polymerase promoter U6, connected with a hairpin structure between sense and antisense sequences of TGF-b1. The siRNA expression mediated by this vector is available to replicate without host immune response and cytotoxicity [18]. To investigate the mechanism of TGF-b1 siRNA in hepatic fibrosis, we examined the expression of TGF-b1, type I collagen, and a-SMA in experimental animals treated with and without TGF-b1 siRNAs. Expression of TGFb1 mRNA and protein was up to 1.5 and 4.5 times higher, respectively, in the HD group. However, when TGF-b1 siRNAs were administered in cirrhotic mice, the up-regulated TGF-b1 level of expression was significantly decreased. Next we observed the expression of type I collagen, which was also significantly increased in the HD group in comparison with the normal group. Administration of TGF-b1 siRNA in cirrhotic mice induced amelioration of fibrotic change. Remodeling of ECM excessively deposited in cirrhotic livers is performed by TGF-b1 siRNA getting inactivated inside the fibrotic liver through observation of a-SMA, which is a key fibrogenic marker in liver cirrhosis [19]. a-SMA has been decreased to enhance liver
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restoration by blocking TGF-b1 signaling using TGF-b1 siRNA. In addition, immunohistochemical stain showed that the expression of TGF-b1 protein was decreased. The expression of a-SMA and type I collagen was also decreased in THD group compared with increased expression of those in the HD group. Body weight maintenance was another important parameter for the effectiveness of our experiments [20]. The body weight of the experimental mice was more or less maintained in the THD group. Furthermore, serum ALT/AST level was decreased in the THD group compared with the HD group, indicating that TGF-b1 siRNA might effectively attenuate liver damage and improve physiological status. In this study, TGF-b1 expression was increased in the cirrhotic model with accumulation of ECM proteins. These proteins were suppressed by TGF-b1 siRNAs in the cirrhotic model as in our study of experimental glomerulonephritis (in press). Considered together, it is suggested that the antifibrotic role of TGF-b1 siRNAs might directly impact the level of TGF-b1 expression affected with expression of ECM components, such as collagen and overall liver fibrogenesis. In conclusion, TGF-b1 siRNA strategy suppresses TGFb1 overexpression for the murine cirrhotic liver model. The suppressive effect of TGF-b1 siRNAs was also applied to the expression of fibrosis-related proteins, such as type I collagen and a-SMA. These findings suggest that TGF-b1 siRNAs may be a useful tool for developing new therapeutic applications for the prevention of liver cirrhosis. Acknowledgment This work was supported by the grant of Research Institute of Medical Science, Catholic University of Daegu (2005). References [1] S.L. Friedman, Liver fibrosis—from bench to bedside, J. Hepatol. 38 (Suppl 1) (2003) S38–S53. [2] A.M. Gressner, R. Weiskirchen, K. Breitkopf, S. Dooley, Roles of TGF-b in hepatic fibrosis, Front Biosci. 7 (2002) d793–d807. [3] D.M. Bissell, Chronic liver injury, TGF-b, and cancer, Exp. Mol. Med. 33 (2001) 179–190. [4] T. Aboul-Fadl, Antisense oligonucleotides: the state of the art, Curr. Med. Chem. 12 (2005) 2193–2214.
[5] M. Sioud, P.O. Iversen, Ribozymes, DNAzymes, and small interfering RNAs as therapeutics, Curr. Drug Targets 6 (2005) 647–653. [6] M. Arias, S. Sauer-Lehnen, J. Treptau, N. Janoschek, I. Theuerkauf, R. Buettner, A.M. Gressner, R. Weiskirchen, Adenoviral expression of a transforming growth factor-beta1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats, BMC Gastroenterol. 3 (2003) 29. [7] E. Bernstein, A.A. Caudy, S.M. Hammond, G.J. Hannon, Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409 (2001) 363–366. [8] D.M. Dykxhoorn, J. Lieberman, The silent revolution: RNA interference as basic biology, research tool, and therapeutic, Annu. Rev. Med. 56 (2005) 401–423. [9] M. Miyagishi, K. Taira, U6 promoter driven siRNAs with four uridine 3 0 overhangs efficiently suppress targeted gene expression in mammalian cells, Nat. Biotechnol. 20 (2002) 497–500. [10] S.J. Reich, J. Fosnot, A. Kuroki, W. Tang, X. Yang, A.M. Maguire, J. Bennett, M.J. Tolentino, Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model, Mol. Vis. 9 (2003) 210–216. [11] X. Zhang, P. Shan, D. Jiang, P.W. Noble, N.G. Abraham, A. Kappas, P.J. Lee, Small interfering RNA targeting heme oxygenase-1 enhances ischemia–reperfusion-induced lung apoptosis, J. Biol. Chem. 279 (2004) 10677–10684. [12] G. Dorn G, S. Patel, G. Wotherspoon, M. Hemmings-Mieszczak, J. Barclay, F.J. Natt, P. Martin, S. Bevan, A. Fox, P. Ganju, W. Wishart, J. Hall, siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32 (2004) e49. [13] Y. Takabatake, Y. Isaka, M. Mizui, H. Kawachi, F. Shimizu, T. Ito, M. Hori, E. Imai, Exploring RNA interference as a therapeutic strategy for renal disease, Gene Ther. 12 (2005) 965–973. [14] T.V. Achenbach, B. Brunner, K. Heermeier, Oligonucleotide-based knockdown technologies: antisense versus RNA interference, Chembiochem 4 (2003) 928–935. [15] A.M. Gressner, R. Weiskirchen, K. Breitkopf, S. Dooley, Role of TGF-b in hepatic fibrosis, Front Biosci. 7 (2002) 793–807. [16] A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391 (1998) 806–811. [17] T. Tuschl, Expanding small RNA interference, Nat. Biotechnol. 20 (2002) 446–448. [18] T.R. Brummelkamp, R. Bernards, R. Agami, A system for stable expression of short interfering RNAs in mammalian cells, Science 296 (2002) 550–553. [19] A. Desmouliere, B. Tuchweber, G. Gabbiani, Role of the myofibroblast differentiation during liver fibrosis, J. Hepatol. 22 (1995) 61–64. [20] T.S. Yeh, Y.P. Ho, S.F. Huang, J.N. Yeh, Y.Y. Jan, M.F. Chen, Thalidomide salvages lethal hepatic necroinflammation and accelerates recovery from cirrhosis in rats, J. Hepatol. 41 (2004) 606–612.
The antifibrotic effect of TGF-b1 siRNAs in murine model of liver cirrhosis Kyung-Hyun Kim a, Hyun-Chul Kim b, Mee-Yul Hwang a, Hoon-Kyu Oh a, Tae-Sung Lee c, Young-Chae Chang a, Ho-Jung Song a, Nam-Hee Won d, Kwan-Kyu Park a,* a
Department of Pathology, Catholic University of Daegu, College of Medicine, 3056-6 Daemyung 4-Dong, Nam-Gu, Daegu 705-718, Republic of Korea b Kidney Institute, Keimyung University School of Medicine, Daegu 700-712, Republic of Korea c Department of Obstetrics and Gynecology, Catholic University of Daegu, College of Medicine, Daegu 705-034, Republic of Korea d Department of Pathology, College of Medicine, Korea Universitiy, 125-1 5-ga Anam-dong Sunguk-gu, Seoul, Republic of Korea Received 10 March 2006 Available online 23 March 2006
Abstract Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-b1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-b1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-b1 siRNAs reduced the expression of TGF-b1 in the livers of CCl4 injection compared with those of control group, and the expression of type I collagen and a-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-b1 siRNAs inhibit TGF-b1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human. 2006 Elsevier Inc. All rights reserved. Keywords: TGF-b1; RNA interference; siRNA; Liver cirrhosis
Liver cirrhosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix (ECM) proteins, which is characteristic of most types of chronic liver diseases [1]. Recent investigations have shown that transforming growth factor (TGF)-b1 is one of the most powerful profibrogenic mediators and plays a major role in the development of liver cirrhosis [2,3]. The pathophysiology of ECM formation during liver cirrhosis is multifaceted and complicated. Therefore, there are numerous attempts to prevent liver cirrhosis including antifibrotic strategies. One of these involves targeting mRNA by using antisense oligodeoxynucleotides (ODNs), which are the sequence-specific binding to target mRNA, resulting in the prevention of gene translation. Also, *
Corresponding author. Fax: +82 53 650 4834. E-mail address: [email protected] (K.-K. Park).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.087
double-stranded ODN (decoy ODN) technology, which has consensus sequence binding of the transcription factor, blocks the activity of a specific transcription factor suppressing gene expression at transcription level. Nevertheless, the greatest limitation of these ODNs is that they are easily degraded by nucleases or by readily nonspecific reaction with the control strand. These problems evoke more nonspecific bindings and adverse effects in vivo [4–6]. To circumvent these problems, we applied another gene silencing method, RNA interference (RNAi), for this study. This is the induction of sequence-specific gene silencing by double-stranded RNA (dsRNA). Long dsRNA molecules are processed to small 21–23 nucleotide interfering RNAs by Dicer, an endogenous RNA III enzyme [7]. Although siRNA has been recently used in mammalian systems to define the functional role of individual genes or diseases [8], in vivo study for liver diseases is not as well characterized as the others.
K.-H. Kim et al. / Biochemical and Biophysical Research Communications 343 (2006) 1072–1078
The aim of this study was to see RNAi phenomena for the prevention of fibrosis in the murine liver cirrhotic model by using siRNAs for TGF-b1. To induce TGF-b1 specific gene silencing, we chose 21-nucleotide complementary sequence with TGF-b1 mRNA ligated into pU6 promoter, which is available to replicate without host immune response and cytotoxicity in vivo. Intraperitoneal CCl4 administration was used for the experiment to induce hepatic fibrosis in the mouse. This study was focussed on the gene silencing and antifibrotic effects of TGF-b1 siRNAs in the murine model of liver cirrhosis. Materials and methods Animal model and protocol. Six-week-old C57BL/6 mice were randomly divided into three groups: a normal group treated nothing (normal control, NC), a hepatic damage group with fibrosis (HD), and a group having hepatic damage with fibrosis treated with TGF-b1 siRNA (THD). The HD group and the THD group received intraperitoneal injection of CCl4 (2 ml/kg) dissolved in corn oil (1:3 ratio) three times a week for 8 weeks. SiRNAs were introduced via tail vein 3 weeks after first CCl4 injection. Mice were sacrificed after every 1 week from siRNA injection and were weighed before sacrifice. All mice were kept at 21-25 C under 12-h dark/light cycles. Construction of TGF-b1 siRNAs. To make TGF-b1 specific gene silencing effect, we chose the complementary 21-nucleotide, 5 0 -AACCAAGGAGACGGAATACAG-3 0 , which does not have crossreactivity with any other genes, has a few secondary structures and has 30–50% of the GC contents. We used the pU6-shX which contains promoter regions of the mouse small nuclear RNA U6 to prolong gene silencing [9]. The selected sequence connected with ttcaagaga 9-mer loop and the end of sequence TTTT, which terminates transcription. This structure cloned into EcoRI, XbaI sites of pU6shX. Transfection of TGF-b siRNAs to the animal and its identification. The deliveries of siRNA were performed through tail vein injection. To confirm the transfection site, the plasmid vector labeled with fluorescence using Mirus (MirusBio, WI, USA). After 24 h of injection, we removed liver from mouse, deep-frozen with OCT compound (Sakura Finetechnical Co., Tokyo, Japan), and sectioned 4 lm thickness and then observed with fluorescence microscopy. Biochemical analysis. Blood samples were obtained from the inferior vena cava. Serum was collected from blood sample by immediate centrifugation at 10,000g for 8 min at 4 C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using automated techniques. Northern analysis. Total RNA was extracted from mouse liver according to the TRI-zol Reagent described previously (Invitrogen, CA, USA). Twenty micrograms of total RNA from each sample was denatured at 65 C for 10 min, electrophoresed on 1.2% agarose/formaldehyde gel containing ethidium bromide, and transferred to a Hybond-N+membrane (Amersham, NJ, USA). The membrane was hybridized with [32P]-labeled cDNA probes in a solution 7% SDS, 1 mM EDTA, 1% BSA, and 0.5 M NaPO4 at 65 C overnight. The membrane was then washed 2 · SSC containing 0.1% SDS at room temperature 1 min, twice with 0.2 · SSC containing 0.1% SDS at 65 C for 30 min. Then AGPA film (Agfa-Gevaert, N.V, Belgium) was exposed to the membrane. The probes were radiolabeled by random priming method using Prime-a-gene labeling system (Promega Corp., WI, USA) in the [32P]dCTP. The probes were used for PCR amplification. The amplified products were purified after resolving in 1% agarose gel using QIA gel extraction kit (Qiagen, CA, USA). Protein isolation and Western blotting. Frozen tissues were homogenized in an IPH buffer (50 mM pH 8.0 Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 100 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 M DTT). After incubation for 30 min on ice, the samples were centrifuged at 12,000 rpm, at 4 C for 30 min and the supernatant was transferred to a
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new tube. Total protein was measured with a Bio-Rad Bradford kit (BioRad Laboratories, CA, USA), then 50 lg of total protein was run on a 15% SDS–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA) which was blocked with 5% skim milk in TBS-T (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. The membrane was probed with the primary antibody for 3 h, washed several times with TBS-T, and then incubated with a horseradish peroxidase (HRPO)-conjugated secondary antibody. Finally, the membrane was washed and developed with an ECL detection system (Amersham, NJ, USA). The primary antibodies used in this study were TGF-b1 (Santa Cruz Biotechnology, CA, USA), a-SMA (Sigma, St. Louis, USA), and type I collagen (Abcam, MA, USA). Histopathological investigation. Small pieces of liver from each lobe were kept in 10% formalin solution. Paraffin blocks were prepared. Crosssection taken from the blocks was stained with Masson’s Trichrome. Immunohistochemical stain. Paraffin-embedded sections were deparaffinized with xylene, dehydrated in decreasing concentrations of ethanol, and then treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. Tissue sections were processed in 10 mM citrate buffer (pH 6.0) and heated to 100 C for 10 min for antigen retrieval. Sections then were incubated with antibodies against TGF-b1 (Santa Cruz Biotechnology, CA, USA), a-SMA (Sigma, St. Louis, USA), and type I collagen (Abcam, MA, USA) for 1 h at 37 C. The sections were then incubated in an EnVision system (DAKO, CA, USA) for 30 min at 37 C with washing in PBS before each incubation. DAB (3,3 0 -diaminobenzidine tetrahydrochloride) was used as the color reagent, and hematoxylin was used as a counterstain. Statistical analysis. The data are presented as means standard errors. The statistical difference between means was determined using Student’s t test; p < 0.05 was considered significant.
Results Construction of TGF-b1 siRNA and confirmation of transfection in mouse liver For the experiment, we designed four kinds of TGF-b1 siRNAs containing the consensus sequence without any open end expression and examined the effect of the sequence specific siRNA on TGF-b1. The described sequence interfered with 40% of the TGF-b1 expression (data not shown) compared with the empty vector, so we used mainly this sequence vector for the experiment. Then we examined the distribution and efficacy of TGF-b1 siRNAs in the mouse liver by FITC-conjugated TGF-b1 siRNAs using Mirus liposome intravenously. After 24 h of injection, the mouse liver was removed and sectioned 4 lm thick. The presence of siRNAs within the liver was assessed by immunofluorescence microscopy. Biochemical and physiological changes in the liver As seen in morphological observation, the HD group was severely damaged with CCl4 administration but the THD group was only a little damaged. The body weights in the HD and THD groups were lower than in the normal group. The serum AST and ALT levels were significantly increased in the HD groups compared with those of the normal groups, but were significantly decreased after TGF-b1 siRNA treatment (Table 1). Therefore, TGF-b1 siRNA treatment was effective in reducing the hepatic damage.
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Table 1 Serum level of AST/ALT and body weight
AST ALT Body wt (g)
NC
4W
6W
8W
HD
THD
HD
THD
HD
THD
57.3 ± 16.3 36.7 ± 12.6 32 ± 1.6
334.6 ± 55.1 784.3 ± 48.6 24 ± 0.8
135.7 ± 38.7 79.3 ± 8.5 27.5 ± 0.4
651.9 ± 94.1 1002.5 ± 126.8 21.3 ± 1.9
161 ± 29.5 176.3 ± 35.6 27.8 ± 0.8
610 ± 50.2 883 ± 49.8 23.3 ± 0.5
149 ± 17.3 137.3 ± 24.3 30 ± 1.0
A normal group treated nothing (normal control, NC), a hepatic damage with fibrosis (HD), and a TGF-b1 siRNA treated with HD groups (THD).
The inhibitory effect of TGF-b1 in the liver after TGF-b1 siRNA treatment The administration of CCl4 results in chronic damage to the liver with accumulation of ECM and increased expres-
sion of TGF-b1. The transcription level of TGF-b1 mRNA was up-regulated in the HD group, but declined in the THD group (Fig. 1A). Similarly, the TGF-b1 protein levels were significantly increased in the HD group compared with those of the normal group, but this increase was
Fig. 1. TGF-b1 mRNA and protein expression after TGF-b1 siRNA injection. Northern blot shows the inhibitory effect on TGF-b1 mRNA in cirrhotic liver after the injection of TGF-b1 siRNA (A). Western blot also shows the inhibitory effect on TGF-b1 protein in cirrhotic liver by TGF-b1 siRNAs (B). (HD group, CCl4 administration only; THD group, CCl4 administration with TGF-b1 siRNAs.)
Fig. 2. Western blot shows the inhibition of type I collagen (A) and a-smooth muscle actin protein (B) expression in cirrhotic mouse liver by TGF-b1 siRNA injection. (HD group, CCl4 administration only; THD group, CCl4 administration with TGF-b1 siRNAs.)
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attenuated by TGF-b1 siRNA treatment in the THD group (Fig. 1B). The expression of type I collagen and a-smooth muscle actin expression after TGF-b1 siRNA treatment Chronic damage to the liver also affects expression of the type I collagen, which is major ECM protein. Western blots shown in Fig. 2A, type I collagen expression was significantly increased in the HD group compared with that in normal group, but it was decreased after TGF-b1 siRNA treatment in the THD group. To determine the liver fibrogenesis, we also observed the expression level of a-smooth muscle actin (a-SMA). Its expression was significantly increased in the HD group compared with the normal group. However, its expression in the THD group was significantly reduced when compared with that in the HD group at 6 and 8 weeks (Fig. 2B). The histopathological finding As shown in Fig. 3, total collagen fibers in the liver were observed by trichrome stain. In HD group, the liver was damaged gradually. Periportal fibrotic bands, which are detected by Masson’s Trichrome stain, were gradually increased, but were much decreased in the
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THD group against with the HD group. There were only minimal residual periportal bands in the THD group. Therefore, TGF-b1 siRNA treatment moderates liver fibrogenesis. Immunohistochemical finding of TGF-b1, type I collagen, and a-SMA In the HD group, the TGF-b1 positive cells were increased in number and size. In addition, the immunohistochemical stain intensity was also increased compared with the THD group. There were few TGF-b1 positive cells in the THD group (Fig. 4). Type I collagen was increased in periportal area in the HD group. However, it was decreased after TGF-b1 siRNA treatment (Fig. 5). a-SMA was also positive in portal vein and periportal region in the HD group, but not in the THD group (Fig. 6). There were significant decreases of fibrosis-related proteins after TGF-b1 siRNA treatment. Discussion In vivo gene silencing studies have been reported in animal models of choroidal neovascularization [10], lung ischemia–reperfusion injury [11], and neuropathic pain [12].
Fig. 3. Trichrome stain in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of total collagen fiber expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, total collagen fiber was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
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Fig. 4. Immunohistochemistry for TGF-b1 in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of TGF-b1 expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, TGF-b1 was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
Fig. 5. Immunohistochemistry for type I collagen in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of type I collagen expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, type I collagen was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
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Fig. 6. Immunohistochemistry for a-SMA in cirrhotic mouse liver. The HD group is a cirrhotic mouse liver (A–C). The THD group is a cirrhotic mouse liver treated with TGF-b1 siRNA plasmid vector (D–F). The time-dependent increase of a-SMA expression was observed in cirrhotic livers (A, 4 weeks; B, 6 weeks; and C, 8 weeks). In contrast, a-SMA was decreased in the THD groups compared with the HD group (D, 4 weeks; E, 6 weeks; and F, 8 weeks) (100·).
Recently, Y Takabatake et al. [13] have reported that TGFb1 siRNAs significantly suppressed TGF-b1 mRNA and protein expression, thereby ameliorating the progression of matrix expansion in experimental glomerulonephritis. To make an animal model of liver cirrhosis in this study, intraperitoneal injection of CCl4 was used for chronic liver damage [14]. The CCl4 injection results in an increased necrosis followed by the development of fibrosis. Many factors involved in this event, especially TGF-b1 is the most potent regulator in liver cirrhosis. It regulates ECM proteins by reducing matrix degradation and stimulating matrix accumulation [3,15]. We therefore applied it as a target factor for this experiment using the RNAi principle. The phenomenon of RNAi was first discovered in Caenorhbditis elegans as a response to double-stranded RNA (dsRNA), which resulted in sequence-specific gene silencing [16]. In contrast to C. elegans, where RNAi effects are stable, long lasting, and passed onto the offspring [3], gene silencing by transfected siRNA duplexes in mammalian cells is transient. This is because mammalian cells lack the RNA-dependent RNA polymerases that amplify siRNAs in C. elegans. To get around this problem, vectorbased systems for the introduction and stable expression of siRNA in target cells have been developed [17]. These vectors contain RNA polymerase III promoters that either express sense and antisense strands from separate promoters (tandem type) or express short hairpin RNAs (shRNAs) that are cleaved by the Dicer to produce siRNA.
Using this technique, RNAi seems to be more suitable for knockdown experiments and interferes less with following functional readouts in comparison with other gene silencing method, such as antisense [14]. To obtain the maximum efficacy when we transfect siRNAs to our animal model, we constructed TGF-b1 siRNAs containing RNA III-dependent RNA polymerase promoter U6, connected with a hairpin structure between sense and antisense sequences of TGF-b1. The siRNA expression mediated by this vector is available to replicate without host immune response and cytotoxicity [18]. To investigate the mechanism of TGF-b1 siRNA in hepatic fibrosis, we examined the expression of TGF-b1, type I collagen, and a-SMA in experimental animals treated with and without TGF-b1 siRNAs. Expression of TGFb1 mRNA and protein was up to 1.5 and 4.5 times higher, respectively, in the HD group. However, when TGF-b1 siRNAs were administered in cirrhotic mice, the up-regulated TGF-b1 level of expression was significantly decreased. Next we observed the expression of type I collagen, which was also significantly increased in the HD group in comparison with the normal group. Administration of TGF-b1 siRNA in cirrhotic mice induced amelioration of fibrotic change. Remodeling of ECM excessively deposited in cirrhotic livers is performed by TGF-b1 siRNA getting inactivated inside the fibrotic liver through observation of a-SMA, which is a key fibrogenic marker in liver cirrhosis [19]. a-SMA has been decreased to enhance liver
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restoration by blocking TGF-b1 signaling using TGF-b1 siRNA. In addition, immunohistochemical stain showed that the expression of TGF-b1 protein was decreased. The expression of a-SMA and type I collagen was also decreased in THD group compared with increased expression of those in the HD group. Body weight maintenance was another important parameter for the effectiveness of our experiments [20]. The body weight of the experimental mice was more or less maintained in the THD group. Furthermore, serum ALT/AST level was decreased in the THD group compared with the HD group, indicating that TGF-b1 siRNA might effectively attenuate liver damage and improve physiological status. In this study, TGF-b1 expression was increased in the cirrhotic model with accumulation of ECM proteins. These proteins were suppressed by TGF-b1 siRNAs in the cirrhotic model as in our study of experimental glomerulonephritis (in press). Considered together, it is suggested that the antifibrotic role of TGF-b1 siRNAs might directly impact the level of TGF-b1 expression affected with expression of ECM components, such as collagen and overall liver fibrogenesis. In conclusion, TGF-b1 siRNA strategy suppresses TGFb1 overexpression for the murine cirrhotic liver model. The suppressive effect of TGF-b1 siRNAs was also applied to the expression of fibrosis-related proteins, such as type I collagen and a-SMA. These findings suggest that TGF-b1 siRNAs may be a useful tool for developing new therapeutic applications for the prevention of liver cirrhosis. Acknowledgment This work was supported by the grant of Research Institute of Medical Science, Catholic University of Daegu (2005). References [1] S.L. Friedman, Liver fibrosis—from bench to bedside, J. Hepatol. 38 (Suppl 1) (2003) S38–S53. [2] A.M. Gressner, R. Weiskirchen, K. Breitkopf, S. Dooley, Roles of TGF-b in hepatic fibrosis, Front Biosci. 7 (2002) d793–d807. [3] D.M. Bissell, Chronic liver injury, TGF-b, and cancer, Exp. Mol. Med. 33 (2001) 179–190. [4] T. Aboul-Fadl, Antisense oligonucleotides: the state of the art, Curr. Med. Chem. 12 (2005) 2193–2214.
[5] M. Sioud, P.O. Iversen, Ribozymes, DNAzymes, and small interfering RNAs as therapeutics, Curr. Drug Targets 6 (2005) 647–653. [6] M. Arias, S. Sauer-Lehnen, J. Treptau, N. Janoschek, I. Theuerkauf, R. Buettner, A.M. Gressner, R. Weiskirchen, Adenoviral expression of a transforming growth factor-beta1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats, BMC Gastroenterol. 3 (2003) 29. [7] E. Bernstein, A.A. Caudy, S.M. Hammond, G.J. Hannon, Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409 (2001) 363–366. [8] D.M. Dykxhoorn, J. Lieberman, The silent revolution: RNA interference as basic biology, research tool, and therapeutic, Annu. Rev. Med. 56 (2005) 401–423. [9] M. Miyagishi, K. Taira, U6 promoter driven siRNAs with four uridine 3 0 overhangs efficiently suppress targeted gene expression in mammalian cells, Nat. Biotechnol. 20 (2002) 497–500. [10] S.J. Reich, J. Fosnot, A. Kuroki, W. Tang, X. Yang, A.M. Maguire, J. Bennett, M.J. Tolentino, Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model, Mol. Vis. 9 (2003) 210–216. [11] X. Zhang, P. Shan, D. Jiang, P.W. Noble, N.G. Abraham, A. Kappas, P.J. Lee, Small interfering RNA targeting heme oxygenase-1 enhances ischemia–reperfusion-induced lung apoptosis, J. Biol. Chem. 279 (2004) 10677–10684. [12] G. Dorn G, S. Patel, G. Wotherspoon, M. Hemmings-Mieszczak, J. Barclay, F.J. Natt, P. Martin, S. Bevan, A. Fox, P. Ganju, W. Wishart, J. Hall, siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32 (2004) e49. [13] Y. Takabatake, Y. Isaka, M. Mizui, H. Kawachi, F. Shimizu, T. Ito, M. Hori, E. Imai, Exploring RNA interference as a therapeutic strategy for renal disease, Gene Ther. 12 (2005) 965–973. [14] T.V. Achenbach, B. Brunner, K. Heermeier, Oligonucleotide-based knockdown technologies: antisense versus RNA interference, Chembiochem 4 (2003) 928–935. [15] A.M. Gressner, R. Weiskirchen, K. Breitkopf, S. Dooley, Role of TGF-b in hepatic fibrosis, Front Biosci. 7 (2002) 793–807. [16] A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391 (1998) 806–811. [17] T. Tuschl, Expanding small RNA interference, Nat. Biotechnol. 20 (2002) 446–448. [18] T.R. Brummelkamp, R. Bernards, R. Agami, A system for stable expression of short interfering RNAs in mammalian cells, Science 296 (2002) 550–553. [19] A. Desmouliere, B. Tuchweber, G. Gabbiani, Role of the myofibroblast differentiation during liver fibrosis, J. Hepatol. 22 (1995) 61–64. [20] T.S. Yeh, Y.P. Ho, S.F. Huang, J.N. Yeh, Y.Y. Jan, M.F. Chen, Thalidomide salvages lethal hepatic necroinflammation and accelerates recovery from cirrhosis in rats, J. Hepatol. 41 (2004) 606–612.