Male Sprague-Dawley (one year-old) rats (weight, 500–600 g) were provided by the Experimental Animal Center of Wenzhou Medical University (Rui'an, China). All rats were housed at 21±1°C, with 55±15% humidity and a dark/light cycle of 12/12 h, and provided access to food and water ad libitum. The experimental protocols were reviewed and approved by the Ethics Committee of Wenzhou Medical University (Rui'an, China).

Fetal bovine serum (FBS) and high-glucose Dulbecco's modified Eagle medium (DMEM) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Lipofectamine^® 2000 and the RevertAid RT Reverse Transcription kit were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The RNApure Total RNA kit was purchased from Aidlab Biotechnologies Co., Ltd. (Beijing, China). Scramble miRNA, miR-21 inhibitor and miR-21 mimics were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). SacI and HindIII restriction enzymes were purchased from New England Biolabs, Inc. (Ipswich, MA), dual-Luciferase Reporter Assay kit and passive lysis buffer from Promega Corporation (Madison, WI, USA), and SYBR-Green real-time PCR Master Mix was obtained from Toyobo Co., Ltd. (Osaka, Japan). Antibodies against Smad7, α-smooth muscle actin (α-SMA) and β-actin were purchased from Oncogene Science Diagnostics (Cambridge, MA, USA), horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and the enhanced chemiluminescence (ECL) system from Thermo Fisher Scientific, Inc.

HSCs were isolated as previously described, with minor modifications (21). Briefly, the rats were anesthetized by intraperitoneal injection of 54 mg/kg sodium pentobarbital. A small piece (100–200 mg) of liver tissue was excised using scissors and digested in 1 ml digestion solution containing 0.5 mg/ml pronase E, 0.6 mg/ml collagenase and 0.1 mg/ml deoxyribonuclease (from the bovine pancreas, Grade II, Boehringer Ingelheim GmbH, Ingelheim, Germany) dissolved in Gey's balanced salt solution (GBSS) for 45 min at 37°C. The cell suspension was centrifuged at 450 × g for 6 min at 4°C. The cell pellet was resuspended in 15 ml GBSS containing 3 g/l bovine serum albumin and mixed with 12.5 ml 28.7% (w/v) of Nycodenz (Nycomed, Zurich, Switzerland) in GBSS without NaCl. The mixture was centrifuged at 1,400 × g for 20 min at 4°C. Then the white, diffuse band overlaying the Nycodenz cushion was gently aspirated, diluted in 40 ml GBSS and centrifuged at 450 × g for 7 min at 4°C. The cell pellet was suspended and cultured in DMEM supplemented with L-glutamine (4 mmol/l), penicillin (100 IU/ml), streptomycin (100 µg/ml) and 10% FBS in a humidified atmosphere of 5% CO₂/95% air at 37°C. The resultant cell suspension was determined, by vitamin A staining, to contain >90% HSCs, and 95% of cells were determined be viable by trypan blue staining.

HSCs were cultured for 3 days, then were considered static HSCs. Activated HSCs were obtained by incubation with 2 ng/ml TGF-β1. For miR transfection, HSCs were seeded (2×10⁴ cells/well) into 24-well plates. At 10 days, 75 nM scramble miRNA, miR-21 inhibitor or miR-21 mimics were transfected into HSCs using Lipofectamine^® 2000. At 48 h, HSCs were washed with phosphate-buffered saline twice and lysed in 100 µl passive lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) for 15 min. Whole cell lysate was collected by centrifugation at 12,000 × g for 10 min.

The pMIR-reporter vector (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to construct a plasmid containing the 3′-UTR of Smad7. Targetscan software (version, 5.1; Whitehead Institute for Biomedical Research, Cambridge, MA, USA) was used for target prediction. The fragments containing the predicted wild (5′-GCUCACACUUUAAUAUAAGCUA-3′, the region targeted by miR-21 is marked in bold) and mutated (5′-GCUCACACUUUAAUAUAUCGAA-3′, the mutated sequences is marked in bold) sites were directly synthesized (Shanghai GenePharma Co., Ltd.), then subcloned into a pMIR-report vector by double digestion with SacI and HindIII. HEK293 cells, obtained from the Central Laboratory, First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China), were cultured in DMEM supplemented with 10% FBS and antibiotics. HEK293 cells (2.0×10⁵) were seeded into six-well plates and cultured for 24 h, then transfected with 700 ng pMIR-reporter plasmids containing the wild or mutated 3′-UTR of Smad7 in combination with 400 nM of miR-21 mimics or scramble miRNA using Lipofectamine^® 2000. After 48 h the cells were collected and luciferase activity was measured by Dual-Luciferase Reporter Assay kit using a TD20/20 Luminometer (Turner Designs, San Jose, CA, USA). Each experiment was performed in triplicate. The results were expressed as relative Renilla luciferase activity, normalized to firefly luciferase activity.

Detection of miR-21 expression by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA from rat HSCs was extracted by using RNApure Total RNA kit following the manufacturer's instructions. The first-strand cDNA was synthesized by stem-loop primers and RevertAid RT Reverse Transcription kit. qPCR of miR-21 was carried out using miR-21 specific primers (Table I) and SYBR-Green real-time PCR Master Mix on an ABI Prism 7500 machine (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The level of U6 mRNA (Table I) was used as an internal control, and relative mRNA expression was calculated from three independent experiments. Thermocycling conditions were as follows: An initial predenaturation step for 5 min at 95°C, followed by 30 cycles of denaturation at 94°C for 45 sec, annealing at 56°C for 45 sec and extension at 72°C for 10 min. The fold-change in miR-21, relative to U6, was determined by the 2^−ΔΔCq method (22).

Total RNA extraction and first-strand synthesis were performed as described above. Specific primers for SMAD7, α-SMA, COLLA1, COLLA2 and GAPDH were designed using Primer Premier software (Table I; version, 5.0; Premier Biosoft, Palo Alto, CA, USA), and synthesized by Takara Biotechnology Co., Ltd. (Dalian, China). PCR reaction conditions were optimized for each set of primers (Table I), and carried out as described above. The threshold cycle (Ct) was determined and the fold-change in SMAD7, α-SMA, COLLA1, COLLA2 mRNA levels were calculated, relative to GAPDH, using Ct values.

Protein concentration was determined using the bicinchoninic acid assay protein assay kit (Biorbyt, Ltd., Cambridge, UK). Whole-cell extracts (60 µg) were fractionated by 8% SDS-PAGE, then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk/TBST (0.2% Tween-20, 20 mmol/l Tris-HC1, 150 mmol/l NaC1, pH 7.4) for 2 h at room temperature, then incubated with the following primary antibodies: Smad7 (Wuhan Boster Biological Technology, Ltd., Wuhan, China; catalog no. BA1399), α-SMA, (Abcam, Cambridge, UK; catalog no. ab4085) and β-actin (Boster Biological Technology; catalog no. BM0627; 1:200 dilution) overnight at 4°C. After three washes in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit antibody (Abcam; catalog no. AB6721; 1:2,000 dilution) for 1 h at room temperature. Following extensive washing in TBST, staining of the blots was detected using the ECL system. (Thermo Fisher Scientific, Inc.). β-actin was used as the loading control. Image Lab version 4.1 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for analysis.

Quantitative data are presented as means ± standard deviation. The difference among different groups of cells was calculated by Student's t-test using SPSS software (version 17.0, Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

TGF-β1 is a profibrogenic cytokine that induces HSCs activation (3,4) and upregulates expression of miR-21 during fibrosis (15). In the present study, the authors first investigated the effect of exogenous TGF-β1 on miR-21 expression in rat primary HSCs. Incubation with 2 ng/ml TGF-β1 was indicated to significantly enhanced miR-21 expression, in a time-dependent manner (P<0.01 or P<0.001). The effect was greatest 7 days following addition of TGF-β1 (Fig. 1A). However, expression of Smad7 mRNA was significantly reduced in the presence of TGF-β1 (P<0.01; Fig. 1B), and cellular content of Smad7 protein was also significantly reduced followings incubation with TGF-β1 (P<0.01; Fig. 1C).

α-SMA is a marker of myofibroblast formation (21). miR-21 mimics were previously reported to promote expression of α-SMA, in addition to proliferation and invasion of the primary stromal cells of phyllodes tumors, whereas miR-21 antisense oligos inhibited these processes (23). To verify the differentiation of HSCs into myofibroblasts following TGF-β1 stimulation, the authors measured the cellular content of α-SMA mRNA and protein following incubation with miR-21 mimics or an inhibitor. In comparison to cells transfected with scramble miRNA, cells transfected with miR-21 mimics contained significantly higher levels of α-SMA mRNA and protein, while miR-21 inhibitor significantly inhibited α-SMA mRNA and protein expression in activated HSCs upon TGF-β1 stimulation (P<0.01; Fig. 2A and B). miR-21 is reported to promote collagen synthesis in high glucose-treated primary cardiac fibroblasts by targeting dual specific phosphatase 8 (24). miR-21 mimics were identified to significantly upregulated expression of COLLA1 and COLLA2 mRNA in activated HSCs (P<0.001), while the miR-21 inhibitor significantly suppressed their expression (P<0.01; Fig. 2C).

miR-21 overexpression was recently reported to contribute to TGF-β1-induced epithelial-to-mesenchymal transition (EMT) by inhibiting Smad7 (20). Moreover, Smad7 overexpression in the liver inhibits phosphorylation of Smad2/3 and collagen I expression (9). Thus, the authors hypothesize that Smad7 may interfere with the process by which miR-21 promotes collagen I expression in activated rat HSCs. Indeed, miR-21 mimics were observed to significantly reduce expression of Smad7 mRNA and protein in activated HSCs (P<0.01), while a miR-21 inhibitor significantly enhanced expression of Smad7 mRNA and protein (P<0.01 or P<0.001; Fig. 3A and B). These results suggested that miR-21 may regulate Smad7 expression in activated HSCs. To further investigate whether Smad7 is a direct target of miR-21, the authors cloned the wild type or mutated 3′-UTR of the Smad7 gene into a pMIR-report vector and overexpressed these plasmids in HEK293 cells. Furthermore, these cells were transfected with miR-21 mimics or scramble miRNA, and compared the Renilla and firefly luciferase activity in these cells. Firefly luciferase luminescence was quite low in these cells, and only elevated in cells transfected with the plasmid containing the wild type 3′-UTR of Smad7 and miR-21 mimics. Higher levels of firefly luciferase luminescence were detected in the cells transfected with plasmids containing the wild type 3′-UTR of Smad7 and miR-21 mimics, and thus the ratio of Renilla to firefly luciferase in this group was significantly lower than in other groups (P<0.01; Fig. 4). These observations indicated that miR-21 directly binds the 3′-UTR of Smad7.