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Introduction

Liver cirrhosis is a common disease clinically and is life-threatening in humans. It features an imbalance in the synthesis and degradation of extracellular matrix (ECM), primarily collagen, leading to metabolic disorders of the whole liver and a series of severe clinical complications. Hepatic fibrosis constitutes an inevitable pathological stage in the transformation from multiple chronic liver diseases to cirrhosis, in which the activation and proliferation of hepatic stellate cells (HSCs) is pivotal (1). HSC activation and the following abnormal expression of ECM are essential to the onset of liver cirrhosis. Transforming growth factor β1 (TGFβ1) functions as a crucial factor in activating HSCs and promoting its expression of ECM. The TGFβ1 signal transduction pathway is important in regulating the expression of ECM in HSCs (2). Therefore, TGFβ1 is one of the significant targets in the prevention and treatment of hepatic fibrosis.

In the present study, the optimal small interfering RNA (siRNA) sequence targeting the TGFβ1 gene was first confirmed, and the corresponding pGreenPuro/TGFβ1 short hairpin (sh)RNA lentiviral vector was constructed and transfected into HSC-T6 cells in vitro to observe the lentiviral vector-mediated effect of shRNA on TGFβ1 gene silencing and on the expression of collagen type 1 α1 (Col1a1) of the ECM, which was anticipated to provide an experimental basis for the prevention and treatment of hepatic fibrosis.

Materials and methods

Materials

The HSC-T6 cell line was obtained from Shanghai Aiyan Biotech (Shanghai, China); synthesis of the oligonucleotide sequences of the three RNA interference (RNAi) sites and the corresponding controls was performed by Guangzhou RiboBio Co., Ltd. (Guangzhou, China); the gel extraction kit and plasmid extraction kit were from Roche Diagnostics (Basel Switzerland); restriction enzymes, T4 DNA ligase, Taq polymerase and the reverse transcription-polymerase chain reaction (RT-PCR) kit were from Takara Bio, Inc. (Otsu, Japan); the pGreenPuro lentiviral vector was from Shanghai Innovation Biotechnology Co., Ltd. (Shanghai, China); Lipofectamine® 2000 was from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA); anti-TGFβ1 antibody and anti-Col1a1 antibody were from Merck Millipore (Darmstadt, Germany); DNA oligosynthesis of the optimal siRNA sequence, and the primer synthesis and sequencing were performed by Shanghai Generay Biotech Co., Ltd. (Shanghai, China).

Designing and screening of the optimal siRNA site of TGFβ1

A total of three RNAi sites of the TGFβ1 gene of rats were selected, according to its CDs sequence (site 1, GTCAACTGTGGAGCAACAC; site 2, GCACCATCCATGACATGAA; site 3, CCGCAACAACGCAATCTAT). The corresponding siRNAs of the three sites were synthesized respectively: Site 1 sense, 5′-GUCAACUGUGGAGCAACACdTdT-3′ and antisense, 5′-dTdTCAGUUGACACCUCGUUGUG-3′; site 2 sense, 5′-GCACCAUCCAUGACAUGAAdTdT-3′ and antisense, 5′-dTdTCGUGGUAGGUACUGUACUU-3′; site 3 sense, 5′-CCGCAACAACGCAAUCUAUdTdT-3′ and antisense, 5′-dTdTGGCGUUGUUGCGUUAGAUA-3′. The three pairs of siRNAs were transfected into HSC-T6 cells, respectively, according to the manufacturer's protocol.

The detection primers of TGFβ1 (NM_021578.2) and GAPDH (AF106860.2) of rats were designed based on their respective gene sequences. The upstream and downstream primers of TGFβ1 were 5′-ACTACGCCAAAGAAGTCACCC-3′ and 5′-TGAGCACTGAAGCGAAAGC-3′, respectively, and the upstream and downstream primers of GAPDH were 5′-TCTACTGGCGTCTTCA-3′ and 5′-TGAGCCCTTCCACGAT-3′, respectively. Total RNA of HSC-T6 cells transfected with each siRNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. With the total RNA of HSC-T6 cells transfected with each siRNA, respectively, and with template and non-transfected HSC-T6 cells as controls, RT-PCR analysis was performed to confirm the optimal siRNA site among the three siRNA sites.

Construction and identification of the TGFβ1 shRNA lentiviral vector

The shRNA targeting the sequence of the optimal siRNA was designed and synthesized as follows: Sense, 5′-GATCCGTCAACTGTGGAGCAACACCTTCCTGTCAGAGTGTTGCTCCACAGTTGACTTTTTG-3′ and antisense, 5′-AATTCAAAAAGTCAACTGTGGAGCAACACTCTGACAGGAAGGTGTTGCTCCACAGTTGACG-3′; BamH and EcoR digestion sites were introduced into the positive-sense and antisense strands, respectively, and annealed to form a double-stranded structure. The annealed oligonucleotide fragment was cloned into the pGreenPuro plasmid to establish the pGreenPuro/TGFβ1 shRNA lentiviral vector, which was then transfected into competent cells. The positive recombinant plasmid DNA was identified by restriction enzyme digestion and sequencing.

Packaging and titer of TGFβ1 shRNA lentiviral recombinants

Trypsin was used to digest the healthy 293T cells (Type Culture Collection of Chinese Academy of Sciences, Shanghai, China), which were in the logarithmic phase, 24 h prior to transfection, and the cells were then diluted with DMEM high-glucose medium (DMEM; HyClone; GE Healthcare, Chicago, IL, USA) containing 10% FBS (catalog no. F2442; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). and, inoculated into a 6-well plate at 5×105 cells/well for incubation at 37°C, 5% CO2 and saturated humidity. When the culture reached a 70–80% density, Lipofectamine® 2000 gene transfection reagent was used to mediate transfection of the pGreenPuro/TGFβ1 shRNA lentiviral plasmid DNA with the right sequence into 293T cells. The medium was renewed at 8 h and cultivation was continued until 48 h under the same conditions. An inverted fluorescence microscope was used to observe the expression of GFP and capture images.

The incubation was continued until 72 h, when the supernatant of each culture was collected for centrifugation at 4,000 × g and 4°C for 10 min to remove cell debris. A filter (0.45 µm) was used for further filtration. Following concentration, the supernatant was stored in aliquots at −70°C.

Titers were detected and calculated using the double dilution method with the following formula: Viral titer = (P × N/100 × V) ×1/DF; in which P represents the GFP-positive cell count, N represents 105, V represents the volume of viral diluent and DF represents the dilution ratio.

Transfection of HSC-T6 cells with the pGreenPuro/TGFβ1 shRNA lentiviral plasmid

Trypsin was used to digest the healthy 293T cells in the logarithmic phase 24 h prior to transfection, following which the cells were then diluted with DMEM high-glucose medium containing 10% FBS and inoculated into a 6-well plate at 5×105 cells/well for incubation at 37°C, 5% CO2 and saturated humidity. When the culture reached a 70–80% density, the pGreenPuro/TGFβ1 shRNA lentiviral solution was added to the medium for transfection at a ratio of 1:50, with HSC-T6 cells transfected with empty vector or non-transfected cells as controls. An inverted fluorescence microscope was used to observe the expression of GFP and capture images 24–48 h later.

Effects of the pGreenPuro/TGFβ1 shRNA lentiviral vector on the protein expression of TGFβ1 and Col1a1 in HSC-T6 cells

At 48 h post-transfection with the pGreenPuro/TGFβ1 shRNA lentiviral vector, the total protein was extracted by western and IP cell lysis solution (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol from the HSC-T6 cells. The protein concentration was measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology), and 20 µg protein was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were soaked in blocking buffer containing a TGFβ1 rabbit polyclonal antibody (1:600; catalog no. sc-146) or a Col1a1 mouse monoclonal antibody (1:500; catalog no. sc-59772) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), respectively, overnight at 4°C. Subsequently, HRP-labeled mouse anti-rabbit secondary antibody (1:400; catalog no. A0208) and rabbit anti-mouse secondary antibody (1:400; catalog no. A0216) (both from Beyotime Institute of Biotechnology, Inc.) were added, respectively, and incubated at room temperature for 1.5 h. Chemiluminescence substrate was added for co-culture for ~7 min at 25°C. Images of the results were then captured using GADPH (MW, 3.7×104) as the internal reference.

Effects of TGFβ1 shRNA on the gene expression of TGFβ1 and COL1 in HSC-T6 cells

The specific detection primers for rat Col1a1 (NM_053304.1) were designed based on its gene sequence: Upstream 5′-TGATGTATGCTTGATCTGTAT-3′ and downstream 5′-CGGGGACCCATTGGACCTGAA-3′. The specific detection primers of rat TGFβ1 and GAPDH were as described above. Total RNA of HSC-T6 cells transfected with TGFβ1 shRNA was used as the template, and the gene expression of TGFβ1 and COL1 were detected using RT-PCR analysis, with empty vector-transfected cells treated as controls and GADPH gene as the internal reference. A total of 1.2 µg RNA from each sample was transcribed into cDNA using the PrimeScript™ RT kit (Takara Bio, Inc.) according to the manufacturer's protocol. Reverse transcription was performed under the following conditions: 30°C for 10 min and 42°C for 30 min to synthesize the first chain of cDNA; 99°C for 5 min to inactivate reverse transcriptase; cooling at 5°C for 5 min, one circle. The RT product (12 µl) was diluted in water to a total volume of 50 µl. cDNA was then amplified by PCR with primers specific to the target TGFβ1 and Col1a1 sequence. PCR was performed under the following conditions: Predenaturation at 95°C for 5 min, denaturation at 94°C for 45 sec, annealing at 57°C for 1 min, extension at 72°C for 1 min (28 circles) and final extension at 72°C for 5 min. The PCR products (5 µl) were subjected to electrophoresis and confirmed using GoldView™ staining.

Results

Screening of the optimal siRNA site in TGFβ1

RT-PCR analysis was used to confirm the optimal siRNA site among the three sites. Site 1 was identified as optimal, with the sequence GTCAACTGTGGAGCAACAC (Fig. 1).

Figure 1. Identification of three target RNA interference sites of TGFβ1 in HSC-T6 cells. Results of electrophoresis are shown. 1, target; 2, target; 3, target; 4, comparison; M, DNA standard (DL 2000). TGFβ1, transforming growth factor β1.

Construction and identification of the TGFβ1 shRNA lentiviral vector

The recombinant pGreenPuro/TGFβ1 shRNA lentiviral vector was digested using BamH1 and EcoRI to produce a 55 bp shRNA fragment, shown by electrophoresis (Fig. 2). Sequencing verified successful construction of the recombinant pGreenPuro/TGFβ1 shRNA lentiviral vector carrying TGFβ1 shRNA.

Figure 2. Construction and identification of the TGFβ1 shRNA lentiviral vector. Slow enzyme digestion of recombinant viral vector pGreenPuro/TGFβ1 shRNA was performed. M1 and M2, DNA standards; 1, pGreenPuro/TGFβ1 shRNA; 2, BamHI and EcoRI digested pGreenPuro/TGFβ1 shRNA. TGFβ1, transforming growth factor β1; shRNA, short hairpin RNA.

Packaging and titer of TGFβ1 shRNA lentiviral recombinants

Lipofectamine® 2000 gene transfection reagent was used to mediate pGreenPuro/TGFβ1 shRNA lentiviral plasmid DNA transfection of 293T cells (Fig. 3A). Following incubation for 48 h, marked GFP expression was observed under the fluorescence microscope (Fig. 3B). The culture showed a viral titer of 7×107 TU/ml via the double dilution method.

Figure 3. Packaging and titer of TGFβ1 shRNA lentiviral recombinants. Under a normal fluorescence microscope (A) 293T cells did not express GFP, whereas (B) 293T cells packaged with recombinant lentivirus pGreenPuro/TGFβ1 shRNA expressed GFP. Scale bar, 200 µm. TGFβ1, transforming growth factor β1; shRNA, short hairpin RNA.

Transfection of HSC-T6 cells with the pGreenPuro/TGFβ1 shRNA lentiviral plasmid

No expression of GFP was detected in the non-transfected HSC-T6 cells (Fig. 4A), whereas marked expression of GFP was observed under the fluorescence microscope in the HSC-T6 cells transfected with the pGreenPuro/TGFβ1 shRNA lentiviral plasmid (Fig. 4B).

Figure 4. Transfection of HSC-T6 cells with the pGreenPuro/TGFβ1 shRNA lentiviral plasmid. Under the microscope (A) uninfected HSC-T6 cells did not express GFP. (B) pGreenPuro/TGFβ1 shRNA lentivirus infected-HSC-T6 cells expressed GFP. Scale bar, 200 µm. TGFβ1, transforming growth factor β1; shRNA, short hairpin RNA.

Effects of the pGreenPuro/TGFβ1 shRNA lentiviral vector on the protein expression of TGFβ1 and Col1a1 in HSC-T6 cells

The results of the western blot analysis showed that the HSC-T6 cells transfected with pGreenPuro/TGFβ1 shRNA lentiviral plasmid produced faintly-stained anti-TGFβ1 and anti-Col1a1 bands at MW 1.25×104 and 1.35×105, respectively, whereas the empty vector-transfected HSC-T6 cells and non-transfected HSC-T6 cells produced intense staining of bands at the two sites (Fig. 5), which indicated that silencing of the TGFβ1 gene in HSC-T6 cells at the RNA level effectively inhibited the expression of Col1a1.

Figure 5. TGFβ1 shRNA affects HSC-T6 cell expression of TGFβ1 and Col1a1. Electrophoresis output shows protein expression levels of TGFβ1 and Col1a1. 1, TGFβ1 shRNA-infected HSC-T6 cells; 2, empty vector-infected HSC-T6 cells; 3, uninfected HSC-T6 cells. TGFβ1, transforming growth factor β1; Col1a1, collagen type I α1; shRNA, short hairpin RNA.

Effects of TGFβ1 shRNA on the gene expression of TGFβ1 and Col1a1 in HSC-T6 cells

With the total RNA of the HSC-T6 cells 48 h following transfection with the pGreenPuro/TGFβ1 shRNA lentiviral plasmid as a template, the RT-PCR assay showed no expression of TGFβ1 and comparatively mild expression of Col1a1 at the mRNA level. With total RNA from the HSC-T6 cells at 48 h post-transfection with the empty plasmid or non-transfected HSC-T6 cells as templates, the results of the RT-PCR assay revealed high expression levels of TGFβ1 and Col1a1 at the mRNA level (Fig. 6). These results indicated that the recombinant pGreenPuro/TGFβ1 shRNA lentiviral plasmid effectively silenced the TGFβ1 gene and downregulated the expression of Col1a1 at the mRNA level.

Figure 6. TGFβ1 shRNA affects HSC-T6 cell gene expression of TGFβ1 and Col1a1. M, DNA standard (DL2000); 1 and 3, empty vector-infected HSC-T6 cells; 2 and 4, TGFβ1 shRNA-infected HSC-T6 cells. TGFβ1, transforming growth factor β1; Col1a1, collagen type I α1; shRNA, short hairpin RNA.

Discussion

Hepatic fibrosis represents a pathological stage in which abnormal hyperplasia of intrahepatic connective tissues is manifested during multiple chronic liver injury repair processes. It features an imbalance in the generation and degradation of ECM, which leads to the excessive deposition of ECM, predominantly Col1a1, and finally results in decreased hepatic function.

HSCs are located in Disse's interspace, close to liver sinusoidal endothelial cells and hepatocytes. There are only a small number of HSCs in the normal liver, representing 5–8% of all hepatocytes. HSCs bind with vitamin A lipid droplets, and engage in vitamin A metabolism and ECM synthesis (3), which contribute to the excessive deposition of ECM, particularly under pathological conditions. Activated HSCs have a leading role in the process of hepatic fibrosis, where they multiply in quantity, exhibit phenotype changes and producing excessive ECM.

Derived from rat primary hepatic stellate cells infected by the T antigen of SV40, the HSC-T6 cell line is an active form of HSCs, which can express and secrete collagen matrix, and is an ideal model for investigating hepatic fibrosis (4).

Myofibroblasts produce abundant collagen fibers during acute or chronic liver injury, primarily types I and III, with the level of Col1a1 markedly increased. Type I and III collagen accumulate excessively in Disse's space and occupies the interspace of liver sinusoidal endothelial cells, which promotes the onset and development of hepatic fibrosis. Col1a1 constitutes a primary stage of ECM in the case of hepatic fibrosis and cirrhosis, and its expression level can be used as an important indicator to reflect the degree of hepatic fibrosis. High levels of Col1a1 synthesis is central in the onset and development of hepatic fibrosis. TGFβ, which is composed of six subtypes: TGFβ1, TGFβ2, TGFβ3, TGFβ1β2, TGFβ4 and TGFβ5, is essential to the physiological and pathological status of the liver (5), and TGFβ1 has the highest contribution to hepatic fibrosis among the multiple coefficient cytokines (6,7). TGFβ1 regulates ECM deposition resulting from tissue injury-mediated physiological response and also pathological reactions. TGFβ1 is predominantly expressed by liver sinusoidal endothelial cells and Kupffer cells, and mild expression is observed in HSCs in the normal liver, which is markedly upregulated in HSCs in the case of liver injury. The changes in TGFβ1 and Col1a1 showed a consistent trend during hepatic fibrosis, as TGFβ1 directly or indirectly activated HSCs to increase the generation of Col1a1-dominating ECM and decrease its degeneration. The synthesis of ECM, particularly Col1a1, can be reduced via inhibiting the generation of TGFβ1 and inhibiting its secretion and amplification processes. Therefore, TGFβ1 functions as an important initiating factor of hepatic fibrosis. It is clear that silencing TGFβ1 can be used as an important strategy in the prevention and treatment of hepatic fibrosis. Inhibiting the expression or biological activities of TGFβ1 via antisense oligonucleotides or drugs can reduce the generation of ECM and the deposition of fibrous protein (8,9), although this is ineffective. However, the increase of RNAi techniques provides novel strategies for reducing ECM generation and fibrous protein deposition via gene silencing.

RNAi refers to sequence-specific posttranscriptional gene silencing in which endogenous or exogenous double-stranded RNA combines with and degrades intracellular mRNA with the homologous sequence (10). The RNAi technique has high specificity, efficiency and stability (11), and is a useful tool with which gene function and interrelation among upstream and downstream molecules in signal transduction systems are investigated. However, this technique is limited by the fact that the transfection of short-chain RNA can only be sustained for a few days in mammalian cells, whereas lentiviral vector techniques can realize longer term gene expression inhibition, which is more appropriate for in vivo experiments (12).

Therefore, the present study selected three RNAi sites in the rat TGFβ1 gene according to its CDs sequence, and the optimal siRNA sequence was confirmed using RT-PCR analysis. Subsequently, shRNA targeting the sequence of the optimal siRNA was designed, synthesized and cloned into the human pGreenPuro plasmid to establish the pGreenPuro/TGFβ1 shRNA lentiviral vector, and HSC-T6 cells were effectively transfected with this recombinant lentiviral vector. RT-PCR and western blot analyses confirmed that the pGreenPuro/TGFβ1 shRNA lentiviral vector effectively mediated the expression of TGFβ1 in the HSC-T6 cells silenced by TGFβ1 shRNA, and simultaneously downregulated the expression of Col1a1. These results indicated that TGFβ1 shRNA downregulated the activating signal of HSCs to reduce the generation of Col1a1-dominating ECM of HSCs, to alleviate or inhibit hepatic fibrosis. Therefore, TGFβ1 shRNA is among the novel strategies for the prevention and treatment of hepatic fibrosis.

The present study was limited by the fact that the expression of Col1a1 was detected only at the mRNA and protein levels following TGF-β interference. The secretion of Col1a1 and changes in other components of the ECM, including collagen type III, also require consideration in the overall situation, requiring further investigation.

Acknowledgements

This study was supported by the National Natural Science Foundation (grant no. 81273918), the Project of Traditional Chinese Medicine Science and Technology of Chongqing (grant no. 2012-2-63) and the Specific Project of Traditional Chinese Medicine of Chinese PLA, Chongqing, China (grant no. 2010ZYZ231).

References