Following approval from the China Medical University Ethics Committee of Shengjing Hospital (Shenyang, China), tissue samples were collected between August 2013 and August 2015 according to the ethical rules for human experimentation stated in the 1975 Declaration of Helsinki. Full-thickness fetal skin tissues were obtained from the lower leg of 5 mid-gestational fetuses (3 males and 2 females; gestational age, 20–23 weeks) and 5 late-gestational fetuses (3 males and 2 females; gestational age, 30–34 weeks). None of the pregnancies were terminated due to miscarriage or elective abortion, and the reason for termination of pregnancy in each case is summarized in Table I. Women undergoing termination of pregnancy provided written informed consent for the use of fetal tissue in the present study. Samples of HS and paired normal skin tissues were collected during plastic surgery procedures. The HS sample patient profiles are summarized in Table II. All patients provided informed consent. All tissues were collected at the Shengjing Hospital of China Medical University. No patients had been diagnosed with a systemic disease or previously treated for scarring. KCs and HSFbs were cultured as described previously (8,9,12). Cell cultures at passage 3–5 were used in all experiments.

For co-culture of HSFbs and fetal KCs, KCs were seeded at a density of 1×10⁵ cells/cm² on Transwell clear polyester membrane inserts with a 0.4-mm pore size (Costar; Corning Incorporated, Corning, NY, USA) in Epilife™ growth medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% human keratinocyte growth supplement (Invitrogen; Thermo Fisher Scientific, Inc.) and grown to confluence at 37°C, 100% humidity and 5% CO₂. The medium was subsequently changed to high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), and the cells were raised to the air-liquid interface. Prior to co-culture, HSFbs were seeded at a density of 10⁵ cells/cm² onto six-well plates in high-glucose DMEM with 10% FBS, incubated for 48 h at 37°C, and subsequently cultured in serum-free medium for an additional 24 h at 37°C. Following washing with PBS, the membranes containing KCs were transferred onto the cultured fibroblasts and maintained at 37°C, in serum-free low calcium KC growth medium supplemented with 5 ng/ml epidermal growth factor for 2 days. HSFbs cultured in isolation with an empty insert served as the control group. All experimental and control samples were produced in triplicate.

TargetScan Human (www.targetscan.org/vert_61) was used to identify the potential targets of miR-940.

miR-940 mimics (cat. no. MSY0004983; Qiagen GmbH, Hilden, Germany), miR-940 inhibitors (cat. no. MIN0004983; Qiagen GmbH) and a negative control duplex (NC; cat. no. MSY0002505; Qiagen GmbH) were employed to investigate the functions of miR-940 in fetal KCs. Fetal KCs (5×10⁵) were seeded in six-well plates (Nest Biotechnology Co., Ltd., Wuxi, China). At 70% confluence, the cells were subjected to 50 mM transfections for 48 h using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol (13).

Fibroblast proliferation was measured using a tetrazolium reagent, 2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1; cat. no. C0036; Cell Counting kit; Beyotime Institute of Biotechnology, Haimen, China) at 48 h after the initiation of co-culture, according to the manufacturer's protocol. The optical density was measured by determining the absorbance at 450 nm. The results are expressed as the ratio compared with the control. Data were collected from three independent experiments, each of which was performed in duplicate.

To detect miRNAs from fetal KCs and fetal epidermis specimens, RNAs were prepared from cells and epidermis using the mirVana miRNA Isolation kit (Ambion; Thermo Fisher Scientific, Inc.). The RT-qPCR for miRNA analysis in fetal KCs was performed using iQ SYBR-Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 3 µg total RNA was reverse-transcribed using a specific looped RT primer for each miRNA with a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). qPCR was performed on an Applied Biosystems 7500 system (Thermo Fisher Scientific, Inc.). U6 was used as an internal control. Total RNA was prepared from fibroblasts at 48 h after the initiation of co-culture using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The fibroblasts were washed three times with PBS prior to extraction. To analyze mRNA expression in fibroblasts, 0.5 µg total RNA was reverse-transcribed using random hexamer primers and Moloney murine leukemia virus reverse transcriptase under the conditions recommended by the manufacturer (Invitrogen; Thermo Fisher Scientific, Inc.). The iQ SYBR-Green Supermix and Applied Biosystems 7500 system were additionally used for qPCR of mRNAs in fibroblasts. GAPDH was used as an internal control. Fold changes of miRNA and mRNA expression were calculated using the 2^−ΔΔCq method (14). The RT-qPCR reaction conditions were 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. Primer sequences are presented in Table III.

Dual luciferase reporter assays were performed as reported previously (15). Fetal mid-gestational KCs were seeded at a density of 5,000 cells/well in 96-well plates and co-transfected with a pMir-Report luciferase vector (1.2 µg) containing a wild-type or mutant form of the 3′ untranslated region of TGF-β1, which contains a miR-940 binding site, pRL-TK Renilla luciferase vector (0.12 µg; Promega Corporation, Madison, WI, USA) and miR-940 mimics (50 mM). At 48 h post-transfection, the luciferase activities were determined with the Dual-Luciferase Reporter Assay System (Promega Corporation). Renilla luciferase activity was used as an internal control. Following normalization to Renilla luciferase activity, the firefly luciferase activity was analyzed. All results are representative of three independent assays.

Total protein was extracted from the indicated cells using a Mammalian Cell Lysis kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol. Cells were washed with ice-cold PBS prior to lysis by mechanical disruption. The protein concentration was determined using the Bradford method. A total of 50 µg proteins mixed in loading buffer were separated on 1 mm NuPage Novex 10% Bis-Tris gels using a NuPage MOPS SDS Buffer kit (Thermo Fisher Scientific, Inc.) followed by electrotransfer to 0.2-mm nitrocellulose membranes (Pall Life Sciences, Port Washington, WI, USA). Nonspecific binding sites were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in PBS for 1 h at room temperature. Membranes were subsequently incubated with primary antibodies against fibronectin (cat. no. ab23750; diluted 1:500; Abcam, Cambridge, MA, USA), collagen 1 (cat. no. 84336; diluted 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) or α-smooth muscle actin (α-SMA, cat. no. 19245; diluted 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Following three washes with PBS containing 0.5% Tween-20, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. NA934; diluted 1:10,000; GE Healthcare Life Sciences, Little Chalfont, UK) at room temperature for 2 h. Signals were visualized with Amersham Enhanced Chemiluminescence Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences). For the protein loading control, blots were stripped and stained for GAPDH using an anti-GAPDH antibody (cat. no. ab8245; diluted 1:2,000; Abcam).

TGF-β1 rescue experiments were performed by the addition of 5 ng/ml human recombinant active TGF-β1 (hraTGF-β1; PeproTech EC Ltd., London, UK) to cultures of 10⁶ KCs that had been transfected with miR-940 mimcs at 37°C for 24 h. Fetal KCs were harvested at 48 h post-transfection. Cell proliferation and TGF-β1 expression were analyzed as described above with a cell proliferation assay and western blotting, respectively.

Data are presented as the mean ± standard deviation. Comparisons between two groups were performed using Student's t-test. Multiple intervention experiments were compared by one-way analysis of variance with Tukey's correction for multiple pairwise comparisons. Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

HSFbs and normal skin fibroblasts (NFbs) were isolated from HS and normal tissues, respectively. In order to compare fibroblasts from HS and normal tissues, cell proliferation and α-SMA expression were examined in HSFbs and NFbs. As presented in Fig. 1A, HSFbs maintained notably increased proliferation rates relative to NFbs on days 3–6. RT-qPCR demonstrated significantly increased expression of α-SMA mRNA in HSFbs compared with NFbs at passages 4 and 5 (Fig. 1B).

Primary KCs were isolated from fetal skin tissue and healthy adult skin tissue. HSFbs were isolated from HS tissue. In order to determine whether fetal epidermal KCs exerted any biological effects on HSFb proliferation, a co-culture model of KCs and fibroblasts was employed (Fig. 2). Quantitative analysis demonstrated that fetal mid-gestational KCs exhibited a more potent suppressive effect on the proliferation of HSFbs at 2 days of culture, compared with late-gestational or adult KCs (Fig. 2A). Fibronectin and collagen are major ECM components. Therefore, the expression of fibronectin and collagen 1 in HSFbs co-cultured with KCs was detected. The data demonstrated that fetal mid-gestational KCs exhibited a more potent suppressive effect on ECM synthesis in HSFbs, compared with late-gestational or adult KCs (Fig. 2B-D). In order to determine whether fetal epidermal KCs exerted any biological effects on HSFb differentiation, α-SMA expression was examined in HSFbs co-cultured with fetal and adult KCs. As presented in Fig. 2B and E, fetal mid-gestational KCs exhibited a more potent suppressive effect on the expression of α-SMA in HSFbs, compared with late-gestational or adult KCs.

In order to investigate the exact mechanism through which fetal KCs inhibit HSFb proliferation, differentiation and ECM synthesis, nextgeneration sequencing was performed in our previous study to measure the expression of miRNA in fetal KCs. It was observed that 110 miRNAs, including 22 novel miRNA candidates, were significantly differentially expressed between mid- and late-gestational fetal KCs (10). Experimental assessment of candidate miRNAs in the present study demonstrated that miR-940 was upregulated in fetal mid-gestational KCs (Fig. 3A). The levels of miR-940 were also detected in 5 human fetal mid-gestational epidermal specimens, 5 late-gestational epidermal specimens and 5 adult epidermal specimens by RT-qPCR. The results demonstrated that miR-940 was upregulated in mid-gestational epidermal specimens (Fig. 3B).

In order to assess the role of miR-940 in the suppressive effects of fetal KCs on HSFb proliferation, cell proliferation was examined in HSFbs co-cultured with fetal KCs, in the presence or absence of miR-940 mimics, miR-940 inhibitors or NC. As presented in Fig. 4A and B, miR-940 expression was confirmed by RT-qPCR. The results of the proliferation analysis demonstrated that transfection of miR-940 mimics enhanced cell proliferation inhibition induced by fetal mid-gestational KCs, whereas transfection of miR-940 inhibitors reduced cell proliferation inhibition induced by fetal mid-gestational KCs (P<0.05; Fig. 4C). In order to assess the role of miR-940 in the suppressive effects of fetal KCs on the expression of fibronectin, collagen 1 and α-SMA in HSFbs, RT-qPCR and western blot analyses were performed in HSFbs co-cultured with fetal KCs, in the presence or absence of miR-940 mimics, miR-940 inhibitors or NC. The data demonstrated that transfection of miR-940 mimics enhanced the inhibition offibronectin, collagen 1 and α-SMA expression in HSFbs induced by fetal mid-gestational KCs, whereas transfection of miR-940 inhibitors led to an upregulation of fibronectin, collagen 1 and α-SMA expression in HSFbs, compared with the NC group (Fig. 4D-G). The results of the present study indicated that miR-940 may act as a positive modulator in the suppressive effects of fetal KCs on HS formation.

To obtain additional insight into the molecular mechanisms of miR-940 in the suppressive effects of fetal KCs on cell proliferation, differentiation, and ECM synthesis of HSFbs, the public miRNA database (www.targetscan.org/vert_61) was used to predict the potential targets of miR-940. Due to a highly-conserved binding site, TGF-β1 was selected for further examination. In order to determine the association between miR-940 and TGF-β1, the protein expression of TGF-β1 was measured in fetal mid-gestational KCs with differential expression of miR-940. TGF-β1 protein levels in fetal mid-gestational KCs were markedly increased following downregulation of miR-940 (Fig. 5A). To confirm whether TGF-β1 is a direct target of miR-940, dual luciferase reporter vectors containing the putative TGF-β1 3′-untranslated region target site for miR-940, which was downstream of the luciferase gene (pMir-TGF-β1-wild-type), and a deletion mutant of 5 bp in the seed region (pMir-TGF-β1-mutant), were constructed. As presented in Fig. 5B, luciferase activity assays demonstrated that miR-940 significantly reducedthe activity of wild-type, although not mutant, reporters in fetal mid-gestational KCs. The results of the present study demonstrated the specificity of miR-940 in targeting TGF-β1.

The present study investigated whether the effect of miR-940 overexpression on the suppressive effects of fetal KCs on cell proliferation, differentiation and ECM synthesis of HSFbs was mediated by TGF-β1. TGF-β1 rescue experiments were performed by the addition of 5 ng/ml hraTGF-β1 to cultures of KCs that had been transfected with miR-940 mimics. As presented in Fig. 6A, the proliferation of HSFbs co-cultured with KCs was assessed by WST-1 assays. TGF-β1 notably decreased the suppressive effects of human fetal KCs on the cell proliferation of HSFbs. In addition, fibronectin, collagen 1 and α-SMA expression in HSFbs co-cultured with KCs in the presence of 5 ng/ml hraTGF-β1 was examined by RT-qPCR analysis and western blotting. TGF-β1 reduced the suppressive effects of human fetal mid-gestational KCs on the expression of fibronectin, collagen 1 and α-SMA (Fig. 6B-E). These results revealed the involvement of TGF-β1 in the suppressive effect of fetal KCs on the proliferation, differentiation and ECM synthesis of HSFbs.

The present study demonstrated that miR-940 mimic transfection enhances fetal mid-gestational KC-mediated suppression of cell proliferation, differentiation and ECM synthesis of HSFbs, at least partially via downregulation of TGF-β1 expression (Fig. 7).