The α-SMA (cat. no. sc-53015), VDR (cat. no. sc-13133) and β-actin antibodies (cat. no. sc-47778) were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Secondary antibodies for goat anti-mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) and goat anti-mouse IgG labeled with Cy3 were sourced from Boster (Hubei, China). 1,25(OH)₂D₃ (cat. no. D1530) and human recombinant TGF-β1 (cat. no. 240-B-002/CF) were obtained from Sigma-Aldrich (St. Louis, MI, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Scrambles, miRNA mimics and inhibitors were from Ambion (Austin, TX, USA). α-SMA, VDR and β-actin primers were produced by BGI-Beijing (Beijing, China).

MRC5 human lung fibroblasts and 293A cells were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). These two types of cells were incubated in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l of glucose supplemented (Gibco, Carlsbad, CA, USA), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS; Gibco) at 37°C in a humidified atmosphere with 5% CO₂. MRC5 cells were cultured to ~80% confluence, serum-starved for 24 h, and were treated for 48 h in 2% FBS medium with an ethanol vehicle or 1,25(OH)₂D₃ (100 nM) in the absence or presence of rhTGF-β1 (10 ng/ml).

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. cDNA was generated from total RNA using PrimeScript RT Master mix (5X) (Takara, Dalian, China), according to the manufacturing instructions. To quantify mRNA expression, SYBR Premix Ex Taq (2X) (Takara) was used. The qPCR thermal cycling protocol was programmed in the CFX96™ Real-Time PCR Detection system (Bio-Rad, Hercules, CA, USA) and consisted of an initial denaturation step at 95°C for 30 sec, followed by 40 cycles of denaturation for 5 sec at 95°C, and annealing and extension for 30 sec at 60°C. A primer pair for the detection of human β-actin was used as the internal control. RT-qPCR primers were based on GenBank published sequences and were as follows: α-SMA sense, 5′-GGC GGT GCT GTC TCT CTA TG-3′ and antisense, 5′-CCC ATC AGG CAA CTC GTA AC-3′; VDR sense, 5′-GGC CGG ACC AGA AGC CTT T-3′ and antisense, 5′-CAG CCT TCA CAG GTC ATA GCA-3′; β-actin sense, 5′-ACT GGA ACG GTG AAG GTG AC-3′ antisense, 5′-GGC ACG AAG GCT CAT CAT-3′.

miRNA extracts were prepared using an mirVana™ miRNA Isolation kit (Ambion) and subsequently reverse transcribed using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's instructions. miRNA expression levels were quantified using a TaqMan Universal Master mix II (2X) (Applied Biosystems), normalized to U6 snRNA. Results are presented as fold changes in gene expression calculated using the ΔΔCt method.

Cells were plated in 60-mm dishes in 10% FBS-DMEM, cultured until nearly 80% confluence and serum-starved for 24 h, and subsequently the cells were treated as indicated. After 48 h culture, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and were subsequently harvested with cell lysis buffer with protease inhibitors [20 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 1% Triton X-100, 1 mmol/l EDTA, 1 mmol/l EGTA, 20 mol/l Na₄P₂O₇, 2 mol/l Na₃VO₄, 0.1% SDS, 10% glycerol, 2 μg/ml leupeptin and 1 mmol/l PMSF]. Upon centrifugation at 13,000 × g for 5 min at 4°C, the supernatants were collected and the total protein concentrations were determined by the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China) following the manufacturer's instructions. SDS-PAGE (10%) gels were prepared and 10–20 μg/lane of cellular proteins was loaded. The resolved proteins were transferred to a polyvinylidene difluoride membrane and incubated with primary antibodies (α-SMA, 1:100 dilution; VDR, 1:50 dilution) following the manufacturer's instructions. Following incubation with HRP-conjugated secondary antibodies (1:1,000 dilution), the blotting bands were visualized with ECL chemiluminescent kit (Thermo Scientific, Rockford, IL, USA) and quantified with a ChemiDoc™ XRS⁺ Imaging System (Bio-Rad).

Fibroblasts growing on cover slides were fixed in 4% paraformaldehyde for 1 h. Following being permeabilized with 0.2% Triton X-100 in Tris-buffered saline (TBS) for 30 min, the cells were blocked in TBS containing 2% bovine serum albumin for 1 h. Cells were subsequently incubated with anti α-SMA antibody (1:50 dilution) at 4°C overnight. Cells were washed 3 times and were incubated at room temperature with Cy3-conjugated goat anti-mouse secondary antibody (1:50 dilution) for 1 h. Following this, cells were counterstained with 4′,6-diamidino-2-phenylindole (Boster) for nuclear staining, 5 min in the dark and were washed 3 times. Negative controls were carried out by omitting the primary antibody. Fluorescent images were captured with a laser scanning confocal microscope (Olympus FV1000; Olympus, Tokyo, Japan).

Transfection was performed with scramble, miR-27b mimic (50 nM) or miR-27b inhibitor (100 nM) (Ambion). All the transfection experiments were conducted with Lipofectamine 2000 (Invitrogen), following the manufacturer's instructions. Cells in medium containing 10% FBS were seeded in each well and incubated at 37°C. The transfection mixture was incubated at room temperature for 10 min, and was added to a 6-well plate. After 24 h, cells were serum-starved for an additional 24 h, following which they were treated with vehicle control, 1,25(OH)₂D₃ or TGF-β1 for 48 h in 2% FBS medium. Cells were subsequently harvested and total RNA, miRNA or proteins were extracted and analyzed by RT-qPCR or western blot analysis.

A synthetic oligonucleotide was inserted at the 3′UTR region of the luciferase reporter gene of the pMIR-target vector. The wild-type and mutant 3′UTR of the VDR genes were amplified by PCR and subcloned in the pMIR-target vectors. The constructs were co-transfected into 293A cells along with scramble (50 nM), miR-27b mimic (50 nM) or miR-27b inhibitor (100 nM) using Lipofectamine 2000, according to the manufacturer's instructions. Luciferase activities were measured with the Dual Luciferase Reporter Assay system (Promega, Madison, WI, USA) 48 h after transfection.

Values are expressed as means ± standard error of the mean derived from at least three independent experiments. Comparison of two groups was made with an unpaired, two-tailed Student's t-test. Comparison of multiple groups was made with a one-way analysis of variance followed by Dunnett or Tukey test. P<0.05 was considered to indicate a statistically significant difference. All the statistical analyses were performed and graphs were plotted using the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

Previous studies have documented that 1,25(OH)₂D₃ opposed the effect of TGF-β1 on mouse lung fibroblasts differentiation, as demonstrated by its ability to inhibit TGF-β1-induced expression of α-SMA (27). Consequently, whether 1,25(OH)₂D₃ could inhibit α-SMA expression of human lung fibroblasts induced by TGF-β1 was examined. Based on previous experiments, the optimum concentration of 1,25(OH)₂D₃ (100 nM) was chosen as the dose for the present experiments. Notably, RT-qPCR and western blot analysis revealed that TGF-β1 significantly upregulated α-SMA expression at the mRNA and protein levels in MRC5 cells; however, 1,25(OH)₂D₃ markedly inhibited this effect (Fig. 1A and C). Confocal immunofluorescence analysis of MRC5 fibroblasts also revealed that TGF-β1 increased levels of α-SMA, and the process was inhibited by 1,25(OH)₂D₃ (Fig. 1D).

Several studies showed cross-talk between the TGF-β and vitamin D signaling pathways (28–31) and TGF-β contributed to the decreased expression of VDR in dermal fibroblasts of healthy volunteers (43). Furthermore, 1,25(OH)₂D₃ had a significant effect in vivo on the TGF-β signaling pathway by altering levels of VDR and Smad3, and subsequently affecting the bioactive of TGF-β1 (44). Based on this evidence, we speculated that VDR may be a negative regulator of fibroblast differentiation induced by TGF-β. TGF-β significantly decreased the protein expression of VDR, and treatment of TGF-β-stimulated fibroblasts with 1,25(OH)₂D₃ effectively upregulated the VDR protein level (Fig. 1C). However, the levels of the VDR transcripts did not change (Fig. 1B).

As TGF-β1 is a critical cytokine in the pathogenesis of human lung fibroblast differentiation, and a previous study reported that miR-27b was downregulated in the lungs of mice that were administered with bleomycin, a widely used animal model of lung fibrosis (45), we hypothesized that miR-27b participates in this process and could be relevant for the inhibitory effect of 1,25(OH)₂D₃ on the differentiation of lung fibroblasts induced by TGF-β1. Therefore, the levels of miR-27b were examined in MRC5 cells treated with the ethanol vehicle or 1,25(OH)₂D₃ in the absence or presence of TGF-β1. In the present study, miR-27b expression levels were significantly higher in MRC5 cells induced by TGF-β1. However, treatment of TGF-β-stimulated fibroblasts with 1,25(OH)₂D₃ effectively decreased miR-27b expression (Fig. 2). These data suggest that miR-27b may have a role in regulating the differentiation phenotype of the pulmonary fibroblasts.

To determine whether miR-27b regulates the differentiation phenotype of the pulmonary fibroblasts, human lung fibroblasts were transfected with scramble, miR-27b mimic or miR-27b inhibitor and evaluated α-SMA levels in these cells. Overexpression of miR-27b markedly increased the baseline levels of the α-SMA transcripts and α-SMA proteins in lung fibroblasts (Fig. 3A-a and c). The miR-27b inhibitor decreased the α-SMA levels in lung fibroblasts (Fig. 3B-a and c). Given that TGF-β1 upregulated miR-27b in lung fibroblasts, these data suggest that miR-27b may mediate TGF-β1-induced α-SMA expression. TGF-β1-treated lung fibroblasts were transfected with miR-27b inhibitor and subsequently evaluated α-SMA levels in the cells. miR-27b inhibitor attenuated TGF-β1-induced α-SMA expression at the mRNA and protein levels in lung fibroblasts (Fig. 3C-a and c). These data indicate that TGF-β1-mediated α-SMA expression requires, at least in part, the induction of miR-27b. In addition, these initial experiments have demonstrated that 1,25(OH)₂D₃ reduced α-SMA expression and downregulated miR-27b expression in human lung fibroblasts induced by TGF-β1. When the miR-27b mimic was transfected into the human lung fibroblasts, the reduced expression of α-SMA protein in 1,25(OH)₂D₃-treated cells was attenuated by the miR-27b mimic (Fig. 3D-a and c).

Given that VDR can be a negative regulator of fibroblast differentiation induced by TGF-β, whether miR-27b regulates VDR gene expression was further investigated. MRC5 cells were transfected with scramble, miR-27b mimic or miR-27b inhibitor, and VDR levels were evaluated in these cells. The miR-27b mimic reduced VDR protein levels in lung fibroblasts (Fig. 3A-c). The miR-27b inhibitor increased the VDR protein levels in these cells (Fig. 3B-c). However, miR-27b had no effect on the levels of the VDR transcripts (Fig. 3A-b and B-b), suggesting that miR-27b may affect the VDR translation, but not the transcripts in lung fibroblasts. These initial experiments have demonstrated that TGF-β1 upregulates miR-27b and decreases VDR protein expression, but 1,25(OH)₂D₃ opposed the above effects of TGF-β1. The reduced expression of VDR protein in TGF-β1-treated cells was attenuated by the miR-27b inhibitor (Fig. 3C-c). Furthermore, the miR-27b mimic resulted in a decrease of VDR protein expression in lung fibroblasts treated with 1,25(OH)₂D₃ (Fig. 3D-c). Similarly, miR-27b had no effect on the levels of the VDR transcripts (Fig. 3C-b and D-b). Taken together, these data suggest that miR-27b promotes the differentiation of human lung fibroblasts by suppressing VDR protein expression.

The aforementioned results demonstrate that miR-27b can regulate VDR expression; however, they do not prove that there is a direct interaction between miR-27b and the mRNA of VDR. To further substantiate that miR-27b targets VDR directly, whether there was a perfect match between the seed sequence of miR-27b and a region in the 3′UTR of VDR was verified. In addition, this binding site is identical among mammals (Fig. 4A). These results suggested that VDR may be regulated by miR-27b, thus this prediction was evaluated. Luciferase reporter constructs were used, incorporating a wild-type or mutant 3′UTR of VDR, in which the sequence corresponding to the seed region was altered (Fig. 4B). The reporter vectors were subsequently co-transfected into 293A cells with scramble, miR-27b mimic or miR-27b mimic/miR-27b inhibitor. The miR-27b mimic significantly decreased luciferase activity and introduction of the miR-27b inhibitor increased luciferase activit (Fig. 4C). The miR-27b target site was subsequently mutated to confirm that miR-27b was binding to this sequence. Of note, the effects of the miR-27b mimic or miR-27b inhibitor were abolished (Fig. 4D). Taken together, the results confirm that miR-27b targets VDR 3′UTR specifically and directly.