HSC-T6 (18), rat hepatic stellate cells were purchased from Yingrun Biotechnologies, Inc. (Hunan, China). HSC-T6 cells were seeded (3×105 cells/well) in six-well plates and cultured with high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained at 37°C in a 5% CO₂ humidified atmosphere. Following a 24-h incubation, cells were cultured in serum-free DMEM for ~12 h and then transfected with a miR-203 mimic, which is an artificial miRNA that enhances the function of miR-203, a miR-203 inhibitor, which is an antisense oligonucleotide for miR-203, or a negative control scramble miRNA. All miRNAs were purchased from Shanghai GenePharma Co., Ltd., (Shanghai, China). Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used as the transfection reagent, according to the manufacturer's protocol. The concentration of miR-203 mimic, miR-203 inhibitor and scramble miRNA that were used for transfection was 0.02 µM, and each step was completed in 3 min at room temperature. The culture medium was replaced 4–6 h post-transfection, the cells were cultured for an additional 48 h and then used for subsequent experiments. The sequences of the miRNAs that were used were as follows: miR-203 mimic, forward 5′-GUGAAAUGUUUAGGACCACUAG-3′, reverse 5′-AGUGGUCCUAAACAUUUCACUU-3′; miR-203 antisense oligonucleotide, 5′-CUAGUGGUCCUAAACAUUUCAC-3′; and scramble miRNA, 5′-CAGUACUUUUGUGUAGUACAA-3′.

The efficiency of HSC-T6 cell transfection was assessed in order to optimize the concentrations of miRNAs and transfection reagent that were used in the present experiments, using transient transfection with scramble miRNAs, including fluorescein amidite (FAM)-labeled and unlabeled miRNAs. FAM-labeled miRNAs emit fluorescence following excitation at 420–485 nm, which can be observed using fluorescence microscopy, whereas unlabeled miRNAs do not. Culture and transfection conditions were similar to the aforementioned.

Total RNA was extracted from HSC-T6 cells (~2×106) using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-203 was reverse transcribed using the following stem-loop RT primer:5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTTGAA-3′. Thermocycling conditions were as follows: denaturation at 42°C for 60 min, extension at 70°C for 10 min, storage at 4°C. qPCR was performed on cDNA using SYBR Green Realtime PCR Master Mix-Plus (Toyobo Co., Ltd., Osaka, Japan) and miR-203-specific qPCR primers (Table I; Shanghai GenePharma Co., Ltd.). The reaction volume was 20 µl [7.2 µl diethyl pyrocarbonate-treated water, 10 µl SYBR Green mix, 0.4 µl (10 µM) of each primer and 2 µl cDNA]. Thermocycling conditions were as follows: Initial 1 cycle at 95°C for 180 sec, followed by 40 cycles at 95°C for 12 sec and at 62°C for 40 sec.

To detect the mRNA expression of collagen type 1, α1 (COL1A1), COL3A1, α-smooth muscle actin (α-SMA) and SMAD3, total RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed on the cDNA using specific primers (Table I; Shanghai GenePharma Co., Ltd.) and SYBR Green Realtime PCR Master Mix-Plus (Toyobo Co., Ltd.). The reaction volume was 20 µl [7.2 µl diethyl pyrocarbonate-treated water, 10 µl SYBR Green mix, 0.4 µl (10 µM) of each primer and 2 µl cDNA]. Thermocycling conditions were as follows: Initial 1 cycle at 95°C for 180 sec, followed by 40 cycles at 95°C for 12 sec and at 62°C for 40 sec. Relative gene expression was quantified using the 2^−ΔΔCq method (19); miR-203 expression was normalized to the expression of U6, whereas COL1A1, COL3A1, α-SMA and SMAD3 expression was normalized to β-actin. Experiments were performed in triplicate. GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) was used for analysis.

HSC-T6 cells (~2×106) were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) at 4°C for 30 min and total protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples were denatured at 100°C for 5 min, and equal amounts of extracted protein samples (20 µg) were separated by 8–10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking against non-specific protein binding using 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 60 min at room temperature, membranes were incubated with the following primary antibodies at 4°C overnight: rabbit monoclonal anti-α-SMA (1:1,000; cat no. ab32575; Abcam, Cambridge, MA, USA), rabbit monoclonal anti-Smad3 (1:1,000; cat no. C67H9; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse monoclonal anti-COL1A1 (1:500; cat no. ab6308; Abcam) and anti-COL3A1 (1:500; cat no. ab6310; Abcam), and rabbit polyclonal anti-GAPDH (1:500; cat no. ab8245; Abcam). Membranes were then washed with 1X TBS containing 0.1% Tween-20 (TBST) 3 times for 10 min and incubated with the following horseradish peroxidase-conjugated AffiniPure secondary antibodies: Goat anti-rabbit (1:8,000; cat no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and goat anti-mouse (1:2,000; cat no. 115-035-003; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. The membranes were washed with TBST 3 times for 10 min and the protein bands were visualized by enhanced chemiluminescence (ECL) using SuperSignal™ West Femto Maximum Sensitivity Substrate for ECL (Thermo Fisher Scientific, Inc.). GAPDH was used as the loading control. Blots were semi-quantified by densitometric analysis using the Image Lab software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

HSC-T6 cells were seeded (~1×104 cells/well) in 96-well plates and transfected with miR-203 mimic, miR-203 inhibitor or scramble miRNA for 48 h, using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cellular proliferation was measured using an MTT assay (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Following transfection, 0.5% MTT (20 µl) was added in each well and cells were incubated at 37°C for 4 h. Subsequently, 150 µmol DMSO were added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm and the optical density (OD) of the samples was calculated. Results were averaged from at least three independent experiments.

Bioinformatics analysis was used to predict potential target genes for miR-203; TargetScan (www.targetscan.org/vert_71/) (20), PicTar (pictar.mdc-berlin.de/) (21) and miRanda (www.microrna.org/microrna/home.do) (22) software were used. Based on the results of the bioinformatics analysis, oligonucleotides (62 bp) of wild-type and mutant SMAD3 3′-untranslated region (UTR), containing the putative miR-203 binding sites, were synthesized by Shanghai GenePharma Co., Ltd. and cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, WI, USA) by double digestion with SacI/XhoI restriction enzymes by Shanghai GenePharma Co., Ltd. The dual-luciferase reporter assay was performed in 293T human embryonic kidney cells obtained from the Central Laboratory of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). The 293T cell line is characterized by high transfection efficiency, with no influence on the expression of target genes; therefore, 293T cells were used for this experiment. Briefly, 293T cells were seeded in 24-well plates at a density of 3×104 cells/well, and incubated for 24 h at 37°C in a 5% CO₂ humidified atmosphere. Subsequently, cells were co-transfected with either miR-203 mimics (20 pmol) or scramble miRNA negative control (20 pmol) and a reporter plasmid (50 ng) containing either wild-type or mutant SMAD3-3′UTR sequences, or a blank reporter plasmid, using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent. Following 48 h transfection at 37°C in a 5% CO₂ humidified atmosphere, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation), and the signal was recorded using the Smart Line TL Tube Luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany). Renilla luciferase was used as the internal control.

The statistical significance of the differences between groups was assessed using a Student's t-test for pair-wise comparisons and a one-way analysis of variance followed by a post hoc Dunnett's test for multiple comparisons. Data are expressed as the mean ± standard deviation of at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA).

To investigate the putative roles of miR-203 in the development of hepatic fibrosis, the expression levels of genes associated with hepatic fibrogenesis were assessed. During chronic liver injury, activated stellate cells have been reported to remodel the ECM and enrich the fibril-forming collagen contents, particularly collagen types I and III (23), whereas α-SMA has been identified as a marker of hepatic fibrogenesis (24). In the present study, HSC-T6 cells were transfected with a miR-203 mimic, a miR-203 inhibitor or with scramble miRNA. Transfection efficiency was assessed using FAM-labeled miRNAs (Fig. 1A-D). RT-qPCR was used to confirm that miR-203 expression was significantly increased in HSC-T6 cells following miR-203 mimic transfection, whereas it was significantly suppressed following transfection with the miR-203 inhibitor (P<0.01 vs. scramble miRNA; Fig. 1E).

RT-qPCR and western blot analyses were used to investigate mRNA and protein expression levels of COL1A1, COL3A1 and α-SMA (Fig. 2). Notably, mRNA expression levels of the fibrosis-associated genes COL1A1, COL3A1 and α-SMA were significantly upregulated in HSC-T6 cells transfected with the miR-203 inhibitor, whereas they were significantly downregulated in miR-203 mimic-transfected cells compared with the scramble group (P<0.01; Fig. 2A and B). Similarly, COL1A1, COL3A1 and α-SMA protein expression levels were increased following transfection with the miR-203 inhibitor and decreased following transfection with mimics, compared with the scramble miRNA control group (P<0.01 and P<0.01, respectively; Fig. 2C and D).

To investigate whether miR-203 may be involved in the regulation of HSC proliferation, the MTT assay was used to assess the effects of miR-203 inhibition and upregulation on the proliferative capabilities of rat HSCs. The results demonstrated that HSC-T6 cell proliferation was significantly increased following transfection with the miR-203 inhibitor (P<0.01), whereas it was significantly suppressed in cells transfected with the miR-203 mimic (P<0.01) compared with the scramble group (Fig. 3). These results suggested that miR-203 may serve a role as a modulator of HSC proliferation.

To investigate the potential mechanisms underlying the implication of miR-203 in HSC activation and proliferation, bioinformatics analysis was performed to identify potential target genes of miR-203. TRPV4 has previously been reported as a direct target gene of miR-203 during HSC activation (17), and the TGF-β/Smad signaling pathway has been demonstrated to serve an important role in hepatic fibrosis (25). Therefore, the present study investigated whether SMAD3 may be a direct target gene for miR-203. RT-qPCR and western blot analyses were used to evaluate SMAD3 mRNA and protein expression levels, and revealed that SMAD3 mRNA and protein expressions in miR-203 mimic-transfected cells were significantly reduced compared with the scramble group (Fig. 4A and B). Conversely, SMAD3 mRNA and protein expression levels were significantly increased in HSC-T6 cells transfected with the miR-203 inhibitor (P<0.01; Fig. 4A and B).

To investigate whether SMAD3 may be a target of miR-203, the 3′-UTR of the SMAD3 mRNA was cloned into a pmirGLO vector (Fig. 4C). A dual-luciferase reporter assay demonstrated that cells co-transfected with the plasmid containing the wild-type SMAD3 3′-UTR and the miR-203 mimic exhibited the lowest luciferase activity (P<0.01 vs. mutant SMAD3; Fig. 4D). These results suggested that SMAD3 may be a direct target gene of miR-203.