Melanoma tissues and their corresponding matched adjacent normal tissues, that were 5 cm away from the tumor tissue, were collected from 15 cases of patients with melanoma who did not receive cutaneous radiotherapy or chemotherapy prior to surgical resection at the Department of Oncology, Qilu Hospital of Shandong University (Qingdao, China) between January 2017 and January 2018. The cohort included 8 males and 7 females, with a median age of 51 years (range, 39–78 years). The diagnosis of melanoma was performed by two experienced pathologists on the basis of hematoxylin and eosin staining of 5-µm sections conducted according to the manufacturer's protocols (cat. no. C0105; Beyotime Institute of Biotechnology) and immunohistochemistry staining performed as previously described (23), following fixation in 4% paraformaldehyde at room temperature for 2 h. Following resection, tumor tissues were flushed with 0.9% saline, and regions with no necrosis and good vitality were selected for experiments, while the normal tissues were similarly collected. Nerves, blood vessels and connective tissues were removed. Then, all tissues were rapidly snap-frozen in liquid nitrogen and stored at −80°C until use.

The present study was performed in strict accordance with the Declaration of Helsinki and approved by the Clinical Management Committee of Hunan First People's Hospital (Changsha, China). Written informed consent for research purposes was obtained from all patients. The information of the patients is presented in Table I.

The melanoma A2058 and HS294T cell lines, skin fibroblast foreskin BJ and CCC-HSF-1 cell lines, epithelial HME1 cells from mammary glands and 293T cells were purchased from American Type Culture Collection. 293T cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), whereas all other cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.), respectively, which were both supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at a temperature of 37°C in a humidified incubator with 95% O₂ and 5% CO₂. HME1 skin cells were used as controls.

Total RNA was obtained from tissues and all cell lines using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Then, the isolated RNA was cleared of contaminating genomic DNA using DNase treatment (Thermo Fisher Scientific, Inc.) and the RT reaction was performed using a PrimeScript™ RT kit (Takara Bio, Inc.) in a PCR thermocycling instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min to obtain cDNA. Finally, the expression levels of MKK3 and miR-21 were detected using PCR amplification with a commercially available SYBR Green qPCR SuperMix kit (Invitrogen; Thermo Fisher Scientific, Inc.) on an ABI PRISM^® 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 32 sec (with data collection at the end of the 60°C step at each cycle), followed by a melting curve. GAPDH and U6 small nuclear RNA were used as the endogenous controls to quantify mRNA and miRNA expression levels, respectively. The fold changes of gene expression levels were calculated using the 2^−∆∆Cq method (24). The primers used are presented in Table II.

Protein lysate preparation from human tissues and cells was performed on ice using RIPA buffer (Beyotime Institute of Biotechnology) comprising 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate, 1% NP-40 and 0.1% SDS, and supplemented with 1 mg/ml aprotinin, sodium 1 mM orthovanadate and 0.1 mg/ml phenylmethylsulfonyl fluoride. Concentrations of protein were assessed with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Then, the proteins (30 µg) were separated on a 12% SDS-PAGE gel and transferred to a 0.22-µm PVDF membrane (EMD Millipore). After blocking the membranes in 5% skimmed milk in TBS with 0.1% Tween-20 (TBST) for 2 h at room temperature, the PVDF membranes were incubated in PBS containing dilutions of rabbit anti-MKK3 (1:1,500; cat. no. ab5428; Abcam) and mouse anti-GAPDH (1:2,000; cat. no. ab9484; Abcam) antibodies at 4°C overnight. Subsequently, the PVDF membranes were washed with TBST buffer three times, and incubated with PBS, including an anti-rabbit (cat. no. BA1056) or anti-mouse (cat. no. BA1050) secondary antibody (Wuhan Boster Biological Technology, Ltd.) conjugated to horseradish peroxidase (1:10,000) for 1 h at room temperature with gentle agitation. Following three washes in TBST, the membranes were incubated with ECL substrates (Beyotime Institute of Biotechnology) for 1 min, and the Amersham Imager 600 system (GE Healthcare Life Sciences) or X-ray films (Kodak) were used to visualize the target proteins. All results were normalized to the expression of GAPDH protein using ImageJ version 1.51 software (National Institutes of Health).

All plasmids (pLC3) used were designed and synthesized by Sangon Biotech Co., Ltd. using sequences purchased from Guangzhou RiboBio Co., Ltd., including miR-21 mimics (cat. no. miR10000076-1-5), miR-21 inhibitor (cat. no. miR20000076-1-5a), NC inhibitor (cat. no. miR1N0000001-2-5), MKK3-overexpression plasmid and MKK3-small interfering (si) RNA (cat. no. siB08711160442-1-5). Empty pLC3 vector was used as a NC for all transfections except inhibitor transfection. A2058 cells plated in 6-well plates at a density of 2×10⁵ cells/well were transfected with the various plasmids (1 ng) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequent experiments were performed 48 h following transfection.

The binding sequences between MKK3 and miR-21 were predicted using TargetScan version 7.2 software (25). For the dual-luciferase reporter assay, 293T cells seeded into 24-well plates were transfected with 200 ng miR-21 mimics, miR-21 inhibitor or the corresponding control vectors along with 50 ng psiCHECK™-2 containing wild-type (WT) or mutant 3′-UTR of MKK3 mRNA inserted downstream of Renilla luciferase (Promega Corporation). Following a 48-h incubation, the cells were harvested and assessed for Renilla and firefly luciferase signals using a dual-luciferase reporter gene detection system, according to the manufacturer's protocol (cat. no. E2920; Promega Corporation).

A2058 cells (1,000 cells/well) were treated with the indicated conditions in 96-well plates. Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's protocol. At 0, 1, 2 and 3 days, 90 µl fresh culture media and 10 µl CCK-8 solution (cat. no. C0038; Beyotime Institute of Biotechnology) were added to each sample. Subsequently, transfected melanoma cells were incubated at 37°C for 2 h. Finally, the absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Transfected A2058 cells (1,000 cells/well) were treated with the indicated conditions in 6-well plates. After 48 h, the cells were trypsinized, gently mixed with 0.45% agar medium mixture and re-seeded on 6-well plates covered with a layer of 0.9% agar in DMEM. After 2 weeks, the colonies were stained with 0.2% crystal violet for 5 min at room temperature, and quantified by colony counting.

A2058 cells were transfected with control or miR-21 mimics, MKK3 expression vector or small interfering RNA in 6-well plates. After 48 h of transfection, 2×10⁵ cells were harvested, centrifuged for 5 min at room temperature within 500 × g, and washed three times with cold PBS. For cell apoptosis analysis, a cell apoptosis detection kit was used, according to the manufacturer's protocol (cat. no. C1062; Beyotime Institute of Biotechnology). Briefly, the harvested cells were suspended in 500 ml binding buffer and incubated at room temperature in the dark for 15 min after being labeled with 5 ml Annexin-V-FITC and 5 ml propidium iodide (PI). For the cell cycle analysis, the cells were first fixed with 70% ethanol at room temperature for 1 h, and then incubated with RNase (50 µg/ml) and stained with PI for 30 min at 4°C in the dark. A flow cytometer and WinMDI version 2 software (BD Biosciences) was used to evaluate apoptosis and the cell cycle. The apoptosis rate was calculated as the percentage of cells in early + late apoptosis.

The migration and invasion of A2058 cells were quantitatively measured using a Transwell chamber assay (Costar; Corning, Inc.). The cells (5×10⁴ cells) treated with indicated conditions were seeded on the upper insert coated without or with 2% Matrigel (Corning, Inc.) in 24-well plates for migration or invasion assays, respectively. Subsequently, the lower chamber was filled with 600 µl DMEM supplemented with 10% FBS as a chemoattractant, followed by an incubation at 37°C with 5% CO₂ for 24 h. Next, the cells were permeated with 100% methanol for 15 min at room temperature, stained with 0.1% crystal violet for 20 min at room temperature, washed with PBS three times and dried at 80°C for 30 min. Non-migrated or non-invaded cells on the inside of the wells were removed with a cotton swab, and the the cells were counted in images captured in five random fields under a light microscope (magnification, ×200; Olympus Corporation).

Statistical calculations were performed using IBM SPSS software, version 23.0 (IBM Corp.). Linear regression analysis of MKK3 and miR-21 expression in melanoma tissue was conducted using Microsoft Excel 2010 (Microsoft Corporation). All cell experiments were independently repeated at least three times and all data are presented as the mean ± standard deviation. Protein and mRNA expression values in individual patient tissues are presented as the mean ± standard deviation of three replicates. Any differential expression between two groups was determined using an independent Student's t-test. Statistical comparisons among multiple groups were performed using Tukey's one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Expression of MKK3 and miR-21 in melanoma was evaluated via RT-qPCR in 15 pairs of human primary melanoma specimens and corresponding adjacent skin. The protein expression levels of MKK3 in the tissues were also assessed using western blot analysis. The results demonstrated that MKK3 mRNA was upregulated and MKK3 protein was overexpressed in melanoma biopsy samples compared with their corresponding adjacent normal tissue samples (Fig. 1A and B). Conversely, the miR-21 expression profile was reversed compared with the MKK3 expression in these tissues, which indicated that miR-21 expression was lower in primary melanoma tissues compared with the matched adjacent healthy tissues. The mRNA expression of miR-21 was also determined (Fig. 1C). Therefore, these findings indicated that MKK3 was upregulated, and miR-21 was downregulated in melanoma.

The expression levels of MKK3 and miR-21 in melanoma cells were further examined. The results demonstrated that the mRNA and protein levels of MKK3 in A2058 cells were significantly increased compared with HS294T melanoma cells and the control skin cells (Fig. 2A and B). It was also observed that miR-21 expression in A2058 cells was significantly decreased compared with that in HS294T melanoma cells and the control skin cells (Fig. 2C). Therefore, these data indicated that MKK3 may be enhanced and miR-21 may be downregulated in A2058 cells, which was similar to the results from the clinical specimens; A2058 cells were selected for the following experiments, as the cell line exhibited the highest MMK3 and lowest miR-21 expression levels.

Based on the inverse expression profiles of MKK3 and miR-21, the underlying interaction between MKK3 and miR-21 required investigation. First, TargetScan predicted that MKK3 was a potential target gene of miR-21 (25). Cells were transfected with plasmids containing the WT and mutant 3′-UTR of MKK3 with miR-21. A luciferase reporter assay was conducted to confirm that miR-21 could directly target the 3′-UTR of MKK3. As presented in Fig. 3A, the forced expression of miR-21 decreased the luciferase activity in 293T cells with the WT plasmid (P<0.05), although no significant change was observed in cells transfected with the mutant plasmid. To further detect whether miR-21 could affect MKK3 expression in melanoma cells, RT-qPCR and western blot analyses were conducted to quantify MKK3 mRNA and protein expression following transfection with miR-21 mimics and miR-21 inhibitor in A2058 cells. It was observed that the upregulation of miR-21 suppressed MKK3 expression at the mRNA and protein levels, whereas the downregulation of miR-21 promoted MKK3 expression at the mRNA and protein levels, compared with NC plasmid (Fig. 3B). In addition, the expression of MKK3 in 15 melanoma tissues was negatively correlated with miR-21 expression (Fig. 3C). Thus, these results collectively suggested that MKK3 may be a direct target of miR-21.

To explore the functions of MKK3 and miR-21 in A2058 cells, miR-21 was overexpressed by transfecting with miR-21 mimics, and MKK3 was overexpressed by transfecting with an MKK3 overexpression vector. MKK3 expression was significantly increased in A2058 cells following transfection with the MKK3 vector; conversely, expression was significantly downregulated following transfection with MKK3 siRNA (Fig. 4A) Initially, the roles of miR-21 and MKK3 in cell growth were investigated; compared with the empty vector NC group, miR-21 overexpression and anti-MKK3 expression markedly inhibited the growth of melanoma cells, as determined by CCK-8 and soft agar assays (Fig. 4B and C). Additionally, it was demonstrated that MKK3 overexpression accelerated the growth of melanoma cells (Fig. 4B and C). Therefore, these data suggested that miR-21 might restrain cell growth and MKK3 might stimulate the cell growth of melanoma cells.

The effects of miR-21 and MKK3 on cell apoptosis and cell cycle distribution were assessed. It was revealed that enhanced expression of miR-21 and reduced expression of MKK3 significantly aggravated the percentage of cells undergoing apoptosis and arrested the cell cycle at the G₁ phase compared with the NC (Fig. 5A and B). Nevertheless, overexpression of MKK3 did not elevate the cell apoptosis percentage or delay the cell cycle in G₁ phase (Fig. 5A and B). Taken together, these results suggested that miR-21 may stimulate melanoma cell apoptosis and contributed to cell cycle arrest in G₁ phase, and MKK3 exerted an opposite effect on the apoptosis and cell cycle of melanoma cells.

As it was observed that the expression of miR-21 and MKK3 were closely associated with melanoma features, it was hypothesized that miR-21 and MKK3 may serve an important role in its capacity to metastasize. To test this hypothesis, a Transwell chamber was used to quantify the changes in cell migration and invasion. It was demonstrated that both miR-21 mimics- and MKK3-siRNA-transfected A2058 cells exhibited significantly reduced migratory and invasive abilities compared with NC cells (both P<0.01; Fig. 6A and B). Nevertheless, MKK3-overexpressing melanoma cells exhibited greater migratory and invasive abilities than the NC cells (both P<0.01; Fig. 6A and B). Taken together, these in vitro findings concluded that miR-21 might inhibit tumor cell migration and invasion, whereas MKK3 might induce tumor cell migration and invasion.