Recombinant human TGF-β protein (active; cat. no. ab50036) and SB203580 (P38 inhibitor; cat. no. ab120162) were obtained from Abcam. The mimic (cat. no. miR10000728-1-5) and inhibitor (cat. no. miR20000728-1-5) of miR-375 and their negative controls [mimic control (MControl, cat. no. miR1N0000001-1-5) and inhibitor control (IControl, cat. no. miR2N0000001-1-5)] were synthesized by Guangzhou RiboBio Co., Ltd. The small interfering RNA (siRNA) against Map2k6 (siMapk2k6; cat. no. sc-35913) was purchased from Santa Cruz Biotechnology, Inc. and scramble control RNA (siRNA, cat. no. sc-37007) was used as the control. Primary antibodies against the following proteins were obtained from Abcam: α-SMA (cat. no. ab32575, 1:1,000 dilution), periostin (cat. no. ab14041, 1:1,000 dilution), β-actin (cat. no. ab8226, 1:1,000 dilution), phosphorylated (p)-Smad3 (cat. no. ab52903, 1:1,000 dilution, total (t)-Smad3 (cat. no. ab40854, 1:1,000 dilution) and MAP2K6 (cat. no. ab33866, 1:1,000 dilution). Anti-p-AKT (cat. no. 4060, 1:1,000 dilution), anti-t-AKT (cat. no. 4691, 1:1,000 dilution), anti-p-P38 (cat. no. 4511, 1:1,000 dilution) and anti-t-P38 (cat. no. 9212, 1:1,000 dilution) were purchased from Cell Signaling Technology, Inc.

CCD-19Lu normal human lung fibroblasts were purchased from the American Type Culture Collection (ATCC) and were cultured in complete Eagle's minimum essential medium (EMEM; ATCC^® 30–2003™) containing 10% heat-inactivated fetal bovine serum (FBS, ATCC^® 30–2021™) in a cellular incubator (5% CO₂, 37°C), as previously described (30). Cells were synchronized in serum-free medium for 12 h after achieving 50–60% confluence, and were then stimulated with 1, 3, 5, 10 and 15 ng/ml TGF-β for 24 h or 10 ng/ml TGF-β for 6, 12, 24, 48 and 72 h, in order to assess the effects of TGF-β stimulation on miR-375 expression. To investigate the role of miR-375 in vitro, cells (3×10⁵/ml) were pre-treated with miR-375 mimic (25 nM), inhibitor (50 nM) or their negative controls (Mcontrol, 25 nM; Icontrol, 50 nM) at 37°C for 4 h using Lipofectamine^® RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Subsequently, the cells were cultured in fresh EMEM supplemented with 10% FBS for an additional 24 h before incubation with TGF-β (10 ng/ml) for another 48 h (31–33). For P38 inhibition, CCD-19Lu cells were incubated with the P38 inhibitor, SB203580 (10 µM; 37°C) at 1 h prior to TGF-β stimulation. MAP2K6 knockdown was performed using siMap2k6 at 48 h before TGF-β stimulation, and the efficiency was verified by reverse transcription-quantitative PCR (RT-qPCR). Briefly, cells (3×10⁵/ml) were transfected with siMap2k6 (50 nM) or siRNA (50 nM) at 37°C for 4 h using Lipofectamine^® RNAiMAX reagent as previously described (34).

Total RNA was extracted from the cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, and was then reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), as previously described (35,36). The expression levels of fibrotic markers, collagen type I α1 (Col1α1), Col3α1, Ctgf and fibronectin (Fn) were quantified with SYBR^® Premix EX Taq™ (Takara Biotechnology Co., Ltd.) and normalized to GAPDH. Quantification of miRNAs was performed using a TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and U6 small nucleolar RNA was used for normalization, as previously described (37). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 2 sec, 60°C for 20 sec and 70°C for 10 sec. The primer sequences were as follows: Col1α1 forward, 5′-GAGGGCCAAGACGAAGACATC-3′ and reverse, 5′-CAGATCACGTCATCGCACAAC-3′; Col3α1 forward, 5′-GGAGCTGGCTACTTCTCGC-3′ and reverse, 5′-GGGAACATCCTCCTTCAACAG-3′; Ctgf forward, 5′-CAGCATGGACGTTCGTCTG-3′ and reverse, 5′-AACCACGGTTTGGTCCTTGG-3′; Fn forward, 5′-CGGTGGCTGTCAGTCAAAG-3′ and reverse, 5′-AAACCTCGGCTTCCTCCATAA-3′; miR-375 forward, 5′-AGTGTCGTCAGAAAGAACGAACGGC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGAGTC-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

CCD-19Lu lung fibroblasts were lysed in RIPA lysis buffer (50 mM Tris-HCl, 0.5% NP-40, 250 mM NaCl, 5 mM EDTA and 50 mM NaF) and protein isolation was performed as previously described (38,39). After quantification using the Rapid Gold BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.), a total of 50 µg proteins were then loaded onto 10% SDS-PAGE gels for separation. Subsequently, the proteins were transferred onto PVDF membranes, which were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the indicated primary antibodies overnight at 4°C. Finally, the proteins were labelled with horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. no. GB23303; Servicebio, Inc.) at room temperature for 1 h and scanned using a ChemiDoc Touch Imaging system (Bio-Rad Laboratories, Inc.) in the presence of a ECL reagent (cat. no. G2020-25ML; Servicebio, Inc.). Data were analyzed using the Image Lab software (v6.0.0 Build 25; Bio-Rad Laboratories, Inc.)

The online database TargetScanHuman (Release v7.2; http://www.targetscan.org/vert_72/) was used for target prediction and analysis of miR-375.

The luciferase reporter assay was performed as described previously (33,40). Briefly, the wild type (WT) 3′-untranslated region (UTR) of Map2k6 containing the putative miR-375 binding site or a mutant (MUT; the seed region of the binding site was mutated) 3′-UTR sequence was cloned into the pGL3 Basic vector (Promega Corporation). Subsequently, the WT or MUT reporter plasmid (200 ng) was co-transfected into cells (3×10⁵/ml) with miR-375 mimic (25 nM) or the negative control using Lipofectamine^® RNAiMAX reagent at 37°C. Cells were then assayed for luciferase activity 36 h post-transfection with the Dual-Light Chemiluminescent Reporter Gene Assay system (Titertek-Berthold).

Quantitative data are presented as the mean ± standard deviation (SD) and were analyzed by SPSS 23.0 software (IBM Corp.). Comparisons between two groups were determined by a unpaired Student's t-test, whereas one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used to compare the differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of miR-375 in the development of myofibroblast transdifferentiation and pulmonary fibrosis, the present study initially detected miR-375 expression in lung fibroblasts after TGF-β stimulation. As depicted in Fig. 1A, TGF-β treatment significantly increased the expression of miR-375 in human lung fibroblasts in a dose-dependent manner. In addition, miR-375 expression was also progressively elevated from 6 to 48 h after TGF-β incubation compared with the PBS control group (Fig. 1B). Moreover, it was found that treatment with 10 ng/ml TGF-β for 48 h was sufficient to trigger optimal miR-375 upregulation in vitro; therefore, this time course and concentration were selected for further experiments (Fig. 1A and B). A chemically modified inhibitor was used to suppress miR-375 expression in cultured cells, and the data revealed that miR-375 was downregulated by 82% in vitro post-transfection with this inhibitor (Fig. 1C). As shown in Fig. 1D, the mRNA expression levels of the fibrotic markers, Col1α1, Col3α1, Ctgf and Fn, were notably upregulated in response to miR-375 inhibition. Increased expression levels of α-SMA and periostin in response to TGF-β are known to be hallmarks of myofibroblast transdifferentiation (41). Consistently, miR-375 inhibition was observed to exacerbate TGF-β-induced upregulation of α-SMA and periostin (Fig. 1E). Overall, it was concluded that miR-375 was upregulated in response to fibrotic stimulation, and that miR-375 inhibition aggravated TGF-β-dependent transdifferentiation of lung fibroblasts.

The present study also determined whether miR-375 activation could prevent lung fibroblast transdifferentiation in the presence of TGF-β, which is of more interest in clinical situations as it is a crucial pathogenic factor during fibrotic remodeling. As shown in Fig. 2A, miR-375 mimic transfection markedly suppressed TGF-β-triggered collagen synthesis in lung fibroblasts, as evidenced by the decreased mRNA expression levels of Col1α1, Col3α1, Ctgf and Fn. The expression levels of α-SMA and periostin were also suppressed following treatment with TGF-β in miR-375 mimic-transfected lung fibroblasts, but not in cells transfected with MControl (Fig. 2B). The results also revealed that transfection with the miR-375 mimic caused a 7.94-fold increase in miR-375 expression in vitro compared with the MControl group (Fig. 2C). Taken together, these data indicated that miR-375 helped to negatively regulate lung fibroblast transdifferentiation, which occurred in response to stimulation with TGF-β.

The present study then attempted to explore the possible mechanism through which miR-375 exerted its beneficial effect on lung fibroblast transdifferentiation. It has previously been reported that the Smad-dependent pathway is the most common signaling axis in the progression of myofibroblast transdifferentiation and pulmonary fibrosis (42). The present study detected the phosphorylation of Smad3, which is known as the crucial node for the Smad-dependent pathway. Notably, it was observed that transfection with the miR-375 mimic did not alter Smad3 phosphorylation in the presence or absence of TGF-β (Fig. 3A and B). Previous studies have suggested that miR-375 may be involved in regulating the AKT pathway, which is a well-established, pro-fibrotic kinase cascade (43,44). As shown in Fig. 3A and B, TGF-β stimulation resulted in elevated levels of AKT phosphorylation; however, the miR-375 mimic did not affect AKT activation. MAPK pathways, particularly the P38 kinase branch have been reported to be essential for pulmonary fibrosis (13); therefore, the present study sought to evaluate the phosphorylation status of P38. As exhibited in Fig. 3C-E, the miR-375 mimic decreased, whereas the miR-375 inhibitor increased TGF-β-elicited P38 phosphorylation. To further corroborate the role of P38, lung fibroblasts were pre-treated with SB203580 to inhibit P38 activity as aforementioned. The data indicated that P38 inhibition abolished the adverse effect of the miR-375 inhibitor on lung fibroblast transdifferentiation, as determined by the decreased mRNA expression levels of fibrotic markers (Fig. 3F). In Fig. 3F, cells in the second group were treated with TGF-β and IControl to ensure that the inhibitor had no off-target effects. These data demonstrated that the P38 pathway may be responsible for the miR-375-mediated anti-fibrotic effect.

The present study finally attempted to clarify how miR-375 regulates the P38 pathway in TGF-β-treated lung fibroblasts. It is well-accepted that miRNAs negatively regulate gene expression via binding to the 3′-UTR of target mRNAs (20). The possible targets for miR-375 were predicted using the TargetScan database, and the study became focused on MAP2K6, which is the key upstream kinase for P38 activation. As shown in Fig. 4A, a putative binding site of miR-375 was found in the 3′-UTR of Map2k6. To further investigate whether MAP2K6 was a direct target of miR-375, a luciferase reporter assay was performed. The results revealed that miR-375 mimic incubation markedly decreased luciferase activity in fibroblasts co-transfected with the WT 3′-UTR of Map2k6; however, no alteration in luciferase activity was detected when the putative binding sequence was mutated (Fig. 4B). Similarly, it was observed that the protein expression levels of MAP2K6 were downregulated by the miR-375 mimic, and upregulated by the miR-375 inhibitor in lung fibroblasts following TGF-β treatment (Fig. 4C and D). To further confirm the role of MAP2K6 in P38 activation by miR-375, MAP2K6 expression was knocked down with siMap2k6 (Fig. 4E). As expected, the upregulation of fibrotic markers induced by miR-375 inhibitor transfection was completely abrogated in Map2k6-deficient lung fibroblasts (Fig. 4F). In Fig. 4F, cells in the second group were treated with TGF-β and IControl, whereas cells in the third group were treated with TGF-β, Inhibitor and siRNA to ensure that the inhibitor had no off-target effects. These findings showed that miR-375 may suppress P38 activation via directly inhibiting MAP2K6.