Keloid tissue samples were obtained from 15 patients, and healthy skin samples were obtained from 5 patients, all admitted to the Department of Dermatology at No. 1 Hospital of China Medical University (Shenyang, China) between June 2016 and June 2017. The diagnosis of keloid samples was confirmed by pathological examination. All protocols were approved by the Ethics Committee of the No. 1 Hospital of China Medical University.

Human keloid tissues (n=8) were collected from patients who underwent surgery at the Center for Plastic Surgery of No. 1 Hospital of China Medical University between June 2016 and June 2017. The skin specimens were washed in PBS containing 100 U/ml penicillin-streptomycin (Beyotime Institute of Biotechnology, Haimen, China) and incubated with 2.5 mg/ml dispase II (Roche Applied Science, Penzberg, Germany) overnight at 4°C. The following day, the specimens were washed with PBS, and the dermis was manually separated from the epidermis, cut into small 1-mm sections and seeded in a culture flask. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cultures were maintained at 37°C in an atmosphere with 5% CO₂. The medium was refreshed three times/week. Cells at passages 3–5 were used for the purposes of this study.

Cell proliferation was determined using a standard MTT assay. In brief, keloid fibroblasts were first seeded at a density of 5×10³ cells/well into 96-well culture plates, and cultured in DMEM containing 10% FBS for 24 h. The medium was removed and replaced with DMEM containing 10% FBS and different concentrations (0, 250, 500, 1,000 or 1,500 nM) of TSA (Enzo Life Sciences, Inc., Farmingdale, NY, USA). Following incubation for a further 24, 48 or 72 h, 20 µl MTT reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added, and the cells were incubated at 37°C for 4 h. A total of 150 µl dimethyl sulfoxide was added to stop the reaction. The effect of TSA on cell viability was determined by measuring the absorbance at 490 nm.

Cells were washed three times with cold PBS and then fixed in ice-cold 70% ethanol at 4°C overnight. The cells were washed with cold PBS and each sample was incubated with 500 µl of propidium iodide (PI; 50 µg/ml; Sigma-Aldrich; Merck KGaA) and 50 µl RNase A (50 µg/ml) at 37°C for 20 min in the dark. Analyses were performed using a BD LSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA, USA) and the percentage of cells in the G0/G1, S or G2/M cell cycle phases was analyzed using ModFit software version 3.0 (BD Biosciences). Experiments were repeated three times.

The number of apoptotic cells was quantified using an Annexin-V-allophycocyanin (APC) Apoptosis Detection kit (cat. no. 88-8007-74; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Early apoptotic cells were defined as Annexin-V-positive and PI-negative cells. Analyses were performed using a BD LSRFortessa Cell Analyzer (BD Biosciences) and FlowJo version 10.0.7 software (FlowJo LLC, Ashland, OR, USA). All experiments were repeated three times.

Dermal tissue or dermal fibroblasts were first crushed on ice and total RNA extracted using the miRNeasy mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. Fibroblasts at passages 3–5 were treated with TSA or transfected with mimics and total RNA extracted using the miRNeasy mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. cDNA was synthesized from RNA using the High Capacity cDNA Reverse Transcription kit or TaqMan MicroRNA Reverse transcription kit (Thermo Fisher Scientific, Inc.). Each RT reaction using the High Capacity cDNA Reverse Transcription kit was performed in a 96 well Thermal Cycler (Applied Biosystems, USA) for 10 min at 25°C, 37°C for 120 min, 85°C for 5 min. Each RT reaction using the TaqMan MicroRNA Reverse transcription kit was performed in a 96 well Thermal Cycler (Applied Biosystems, USA) for 30 min at 16°C, 42°C for 30 min, 85°C for 5 min. Primer sequences are listed in Table I. Realtime PCR of mRNA was performed using GoTaq qPCR Master Mix reagent (Promega, WI, USA) and a 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) with following program: Stage 1, 95°C for 2 min; Stage 2, 40 cycles of 95°C for 15 sec, 60°C for 1 min. Analysis of miRNA expression by qPCR was performed using TaqMan miRNA assays (Thermo Fisher Scientific, Inc.) and a 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) with following program: Hold 1, 50°C for 2 min; Hold 2, 95°C for 10 min; Hold 3, 40 cycles of 95°C for 15 sec, then 60°C for 1 min. Relative expression was calculated using the 2^−ΔΔCq method or ΔCq method (15). Confirmation of TSA-induced miRNA clusters in keloid fibroblasts were normalized to the control gene U6 small nuclear RNA and analysis of relative gene Expression data performed with the 2^−ΔΔCq method. Expression of miR-30a-5p in keloid tissue as determined by use of RT-qPCR. miR-30a-5p PCR data were compared with small nucleolar RNA, C/D box 48 and analysis of relative gene expression data performed using the 2^−ΔΔCq method.

Cells were first washed with ice-cold PBS, and lysed with phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). Following centrifugation at 5,000 × g for 15 min at 4°C, the protein concentration was determined using a bicinchoninic acid protein assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Protein lysates (50 µg) were loaded onto 10% SDS-PAGE gels before being transferred to polyvinylidene difluoride membranes. The membranes were blocked with TBS with Tween 20 and 5% non-fat milk for 1 h at room temperature, and subsequently incubated with collagen I antibody (cat. no. BA0325; dilution, 1:200; Wuhan Boster Biological Technology, Ltd., Wuhan, China), BCL2 antibody (cat. no. ab196495; dilution, 1:1,000; Abcam, Cambridge, MA, USA), GAPDH (cat. no. 10494-1-AP; dilution, 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) or β-actin (cat. no. ab90641; dilution, 1:5,000; Abcam) overnight at 4°C. The membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. A0181; dilution, 1:1,000; Beyotime Institute of Biotechnology) at room temperature for 1 h, and detected using enhanced chemiluminescence (Amersham^™ ECL^™ Prime Western Blotting Detection Reagent; GE Healthcare Life Sciences, Little Chalfont, UK) and a MF-Chemi BIS 2.0 Imaging Systems(NDR Bio Imaging Systems, Israel) the following day. Grayscale value analysis of protein was performed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The ratio of the target protein to the internal reference protein was calculated for each group.

All oligonucleotides were synthesized by Ambion (Thermo Fisher Scientific, Inc.). Transfections were performed using Lipofectamine^® RNAiMAX (cat. no. 13778-075; Thermo Fisher Scientific, Inc.). Cells in the growth phase were seeded at 2×10⁵ cells/well in six-well plates. To assess the transfection efficiency of miR-30a-5p mimics, cells were transfected with 40, 60 or 80 nM mimics for 24, 48 or 72 h, respectively. Transfections of 60 nM miR-30a-5p mimics or negative control mimics for 72 h was selected for all experiments.

The miRNA array was performed using three different paired samples. RNA was first reverse transcribed using the TaqMan miRNA Reverse Transcription kit and TaqMan miRNA Multiplex RT assays, respectively; Thermo Fisher Scientific, Inc.) with following program: Stage 1, 40 cycles of 16°C for 2 min, 42°C for 1 min, 50°C for 1 sec; Stage 2, 85°C for 5 min. Expression was profiled using a TaqMan Human MicroRNA array card A, B (V2.1 and V3.0, respectively) and a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.), according to the manufacturer's recommended protocol. Briefly, the array was loaded and run using the 384-well Taqman Low Density Array default thermal-cycling conditions with following program: Stage 1, 94.5°C for 10 min; Stage 2, 40 cycles of 97°C for 30 sec, 59.7°C for 1 min on a 7900HT Fast Real-Time PCR System. The quantification cycle (Cq) values were obtained with 900HT System Fast Real-Time PCR SDS software version 2.3 (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the data were analyzed using RQ manager 1.2 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 small nuclear RNA was selected as a control. The -ΔCq[-(Cq-Cq_U6)] values were calculated, and heatmap analyses were performed with hierarchical clustering (16).

The predicted (TargetScan Human 7.1 and miRanda) or confirmed gene targets of these miRNAs were provided. A dual-luciferase reporter assay was performed using 293T cells. Briefly, the putative miR-30a-5-p binding sites within the 3′-UTR of the BCL2 gene were amplified and cloned into the GV272 vector (GeneChem, Inc., Shanghai, China), and the miR-30a-5p gene was amplified and cloned into the GV251 vector (Shanghai GeneChem, Inc.). Cells (1×10⁵) were seeded in 24-well plates and co-transfected with 100 ng wild-type or mutated BCL2 3′-UTR constructs and 400 ng negative control or miR-30a-5p plasmids using X-tremegene HP (Roche Diagnostics, Basel, Switzerland for ≤48 h. Luciferase activity was determined using the Dual-luciferase Reporter Assay System (cat. no. E1910; Thermo Fisher Scientific, Inc.) following transfection for 48 h. Briefly, Dual-Glo^® Luciferase Assay Reagent was added to the plate, the firefly luminescence measured, Dual-Glo^® Stop & Glo^® Reagent added to the plate and Renilla luminescence measured. The ratio of firefly:Renilla luminescence for each well was calculated. The sample well ratio to the ratio from control wells was normalized. Since the miRNA functions primarily by targeting the 3′-UTR of the target gene, this region may be cloned into a luciferase vector and positioned before the luciferase reporter gene. Luciferase activity in mimic or negative control-transfected cells was subsequently measured. Modifications in gene expression are reflected in the change in luciferase activity, and may quantitatively reflect the inhibitory effect of miRNA on the target gene. With TRAF6-3′UTR as a positive control, the expression of luciferase in the group was significantly decreased (P<0.05), indicating that there was no problem in the whole transfection detection system.

The results are expressed as the mean ± standard deviation of at least three separate experiments, each performed in triplicate. Differences between groups were analyzed using a two-tailed Student's t-test, Mann-Whitney U test or one-way analysis of variance with Tukeys post hoc test using the software GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

An initial dose titration was performed to determine the appropriate concentration of TSA to be used in subsequent experiments. Keloid fibroblasts were treated with media containing increasing doses (0, 250, 500, 1,000 and 1,500 nM) of TSA. The effects of TSA on cell viability were monitored using an MTT proliferation assay. As presented in Fig. 1, keloid fibroblasts treated with TSA demonstrated a statistically significant reduction in cell growth following incubation with TSA for 24, 48 or 72 h. Accordingly, TSA inhibited keloid fibroblast growth in a time- and dose-dependent manner. The proliferation of keloid fibroblasts treated with either 1,000 or 1,500 nM TSA was significantly inhibited and morphological alterations were observed when compared with the controls. Overall, cells treated with 1,000 nM TSA tolerated the treatment well, and preserved their viability compared with the control. Therefore, 1,000 nM TSA was used as the working dose for all subsequent experiments.

To investigate the effects of TSA on apoptosis, cultured keloid fibroblasts were incubated with 1,000 nM TSA for 24, 48 or 72 h, and Annexin V-APC/PI staining for apoptosis detection was performed, followed by flow cytometry. The results demonstrated that keloid fibroblast apoptosis was upregulated following TSA treatment at all three time points tested (P<0.01; Fig. 2). In addition, the apoptosis rate in cells pretreated with TSA for 72 h was increased by 12.5% relative to the controls.

The results demonstrating the impact of TSA treatment on keloid fibroblast proliferation prompted the investigation of the role of cell cycle progression in this phenotype. Following a 24 h incubation period, the average percentage of cells in the G2/M phase increased from 23.0% in the control group to 26.8% in the 1,000 nM TSA group (P=0.5287; Fig. 3). Notably, G2/M arrest was observed following longer incubation periods. After a 48 h incubation period, the average percentage of cells in the G2/M phase increased from 13.2% in the control group to 20.8% in the 1,000 nM TSA group (P=0.0064), while 72 h of TSA incubation was associated with an average increase in the percentage of cells in the G2/M phase from 16.2% in the control group to 33.0% in the 1,000 nM TSA group (P=0.0381; Fig. 3B). These results suggested that TSA may influence the cell cycle of keloid fibroblasts.

The ability of TSA to modulate the expression of collagen type Iα 1 chain (COL1A1) in keloid fibroblasts was investigated by RT-qPCR (Fig. 4A). COL1A1 expression, as detected using western blotting, exhibited an apparent decrease (Fig. 4B). In addition, the protein expression levels of COL1A1 and BCL2 in TSA-treated keloid fibroblasts were seemingly reduced compared with the controls.

It has been previously reported that the expression of specific miRNAs is significantly altered following TSA treatment (17). The altered expression of specific miRNAs has been associated with ECM synthesis, fibroblast differentiation, epithelial-mesenchymal transition (EMT), in addition to cancer initiation, invasion and metastasis (17,18). Therefore, miRNA microarray analysis of keloid fibroblasts from three biological samples treated with 1,000 nM TSA for 72 h was performed in the present study. As illustrated in the heatmap presented in Fig. 5, a number of alterations in miRNA expression were observed. Specifically, of the miRNAs significantly altered by TSA treatment, 22 were upregulated (Table II) and 10 were downregulated (Table III). The predicted (TargetScan Human 7.1 and miRanda) or confirmed gene targets of these miRNAs are provided. Of particular relevance to the present study, the miR-125a and miR-17 sequences, which have been associated with fibroblast proliferation, were observed to be downregulated, while miR-30a-5p and miR-146a, which have been associated with transforming growth factor (TGF)-β signaling, were upregulated. Considering previous studies, a number of miRNAs were selected and alterations in their expression between treatment and control were evaluated by RT-qPCR. The miRNA microarray and RT-qPCR results verified the aforementioned observations, demonstrating that that miR-30a-5p exhibited a marked alteration during TSA-mediated inhibition of keloid fibroblast cell growth (Figs. 6 and 7). It has been reported that miR-30a-5p targets BCL2 mRNA and induces apoptosis in non-small cell lung cancer cells (19,20). Although the change observed was not the most evident, it was found that potential targets of this miR-30a-5p were more pertinent to the aims of the present study. Therefore, the role of miR-30a-5p in keloid fibroblast regulation was further investigated.

Based on the observation that TSA altered the expression of miR-30a-5p in keloid fibroblasts, the expression of miR-30a-5p in keloid tissues was investigated. To achieve this, RT-qPCR was used to examine the expression levels of miR-30a-5p in five healthy skin and seven keloid tissue samples. As exhibited in Fig. 8, the mean expression levels of miR-30a-5p in keloid tissue samples were significantly decreased when compared with the healthy skin samples (P=0.0480). These results suggested that miR-30a-5p may serve an important role in inhibiting the pathological process of keloid formation.

The results presented thus far suggested that miR-30a-5p may be associated with fibroblast proliferation and ECM deposition. The 3′-UTR of BCL2 was predicted to contain a miR-30a-5p binding site (Fig. 9A). To experimentally validate this miRNA-mRNA interaction, a luciferase assay was performed. As presented in Fig. 9B, miR-30a-5p significantly decreased BCL2 3′-UTR reporter activity when compared with the BCL2 3′-UTR negative control group. The inhibitory effect of miR-30a-5p was eliminated when the predicted miR-30a-5p binding site was mutated. TRAF6-3′ UTR was used as a positive control; the expression of luciferase in the group was significantly decreased (P<0.05), indicating that there was no problem in the transfection detection system.

To further investigate the role of miR-30a-5p in keloid fibroblasts treated with TSA, the transfection efficiency of miR-30a-5p mimics within keloid fibroblasts cultured for 72 h was determined. The effect of miR-30a-5p mimics on cell viability was monitored using MTT proliferation assays. The results demonstrated that the proliferation of cultured keloid fibroblasts was reduced by 8% when compared with the controls (P=0.0187; Fig. 10A). To study the inhibitory effect of miR-30a-5p on apoptosis, keloid fibroblast cultures were incubated with 60 nM miR-30a-5p for 72 h, and the level of apoptosis was ascertained using Annexin V-APC/PI staining and flow cytometry analysis. The results demonstrated that keloid fibroblast apoptosis was increased by 5.9% relative to the control (P=0.0031; Fig. 10B and C). However, no effects on cell cycle progression were observed (P=0.6303; Fig. 10D and E).

To investigate potential interactions between the predicted target gene, BCL2, and miR-30a-5p, total RNA and protein from keloid fibroblasts transfected with negative control or miR-30a-5p mimics for 72 h was determined by RT-qPCR. Furthermore, western blot analyses were performed to determine the mRNA and protein expression levels of BCL2 and COL1A1. The mRNA and protein levels of BCL2 (Fig. 11A-C) and COL1A1 (Fig. 11D-E) were significantly decreased in keloid fibroblasts. When compared with the negative control mimic-transfected cells, BCL2 mRNA levels were decreased by 0.73-fold (P=0.0116) and COL1A1 mRNA by 0.68-fold (P=0.0118), and statistically significant reductions in BCL2 (P=0.0361) and COL1A1 (P=0.0012) protein expression levels were also observed.