A375 cells (Chinese Academy of Sciences Cell Bank, Beijing, China) were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and maintained at 37°C in a humidified incubator with 5% CO₂, unless otherwise indicated.

The sequence of miR-183 is 5′-GUGAAUUACCGAAGGGCCAUAA-3′. MiR-183 overexpression and knockdown in A375 human melanoma cells was achieved using an miR-183 mimic, an miRNA mimic negative control (NC) [an insignificant short nucleotide sequence], an miR-183 inhibitor, an miRNA inhibitor NC and blank plasmids were all obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China) following the manufacturer's protocol. Following transfection miR-183 (250 µg/ml) for 12 h by Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, total RNA from each cell group was isolated via TRIzol-chloroform-isopropanol (TaKaRa, Bio, Inc., Otsu, Japan) extraction and miR-183 mRNA expression was assessed using RT-qPCR analysis with an All-in-One™ miRNA RT-qPCR detection kit (GeneCopoeia, Inc., Rockville, MD, USA) following the manufacturer's protocol. The sequences of miR-183 primers, forward: 5′-CGCGGTATGGCACTGGTAGA-3′, and reverse: 5′-AGTGCAGGGTCCGAGGTATTC-3′. MiR-183 expression levels were normalized to levels of U6 snRNA (5′-AAATTCGTGAAGCGTTCC-3′) using the 2^−ΔΔCq method (52). Detection was achieved by SYBR green qPCR with the following conditions: 95°C for 10 min followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 20 sec.

A375 cells transfected with the miR-183 mimic, mimic NC, miR-183 inhibitor or inhibitor NC plasmid were cultured (37°C) overnight into confluent monolayers. Scratches were generated gently in each cell culture using a sterile 200 µl pipette tip. Cell migration towards the mid-line of the scratch was monitored at 0 and 24 h using a light microscope at ×100 magnification.

Following pre-culture in serum-free RPMI-1640 medium for 24 h at 37°C, A375 cells with the aforementioned transfections were trypsinized, re-suspended in serum-free RPMI-1640 medium and diluted to a density of ~10,600 cells/ml. Cells (100 µl of each group) were then seeded in the inner chamber membrane of the Transwell apparatus. For the assessment of invasion activity, the inner chamber was coated with fibronectin under serum-free conditions and the uncoated inner chamber was used for the evaluation of migration activity. RPMI-1640 medium supplemented with 50% fetal bovine serum was added in the lower chamber of the Transwell apparatus in each group and cells were cultured for 24 h at 37°C in a humidified incubator with 5% CO₂. A549 cells on the lower surface of the inner chamber were then gently rinsed with PBS and stained with crystal violet staining solution at room temperature for 10 min (Beyotime Institute of Biotechnology, Haimen, China) prior to being counted using light microscopy at ×200 magnification. Five randomly selected fields of view were selected.

A375 cells that underwent the aforementioned transfections were cultured in 6-well plates under normal culture conditions (37°C for 24 h). A375 cells were homogenized in lysis buffer [10 mM Tris base, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, and a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.)]. Protein concentrations were determined using a BCA assay kit (Thermo Fisher Scientific, Inc.) with bovine serum albumin (2 mg/ml) as a standard. The total protein of each cell group (20 µl) was separated using polyacrylamide gel (15%) electrophoresis under reducing conditions, followed by transferral onto nitrocellulose membranes. Following blocking at 4°C overnight with non-fat milk and preparation with PBS, membranes were probed with antibodies (incubated 16 h at 4°C) targeting human Ezrin (cat. no. ab40839; Abcam, Cambridge, UK; dilution of 1:2,000) and MMP-9 (cat. no. ab76003; Abcam; dilution of 1:1,000), with β-actin (cat. no. ab8227; Abcam; dilution of 1:2,000) as an internal reference. The membranes were then incubated with horseradish peroxidase conjugated secondary antibodies (dilution of 1:5,000, incubated 1 h at room temperature) (Goat Anti-Rabbit IgG H&L; cat. no. ab6702; Abcam) and colorized using an ECL substrate (Thermo Fisher Scientific, Inc.). The image of each sample was captured using chemo-fluorescence-sensitive film and the gray value of each band was analyzed. The quantity of target protein was evaluated by the ratio of its gray value to the internal reference using ImageJ software, version 1.48 (National Institutes of Health, Bethesda, MD, USA).

All experiments were independently repeated at least three times. Statistical analysis was performed using SPSS statistical software, version 17.0 (SPSS, Inc., Chicago, IL, USA). All statistical data were presented as the mean ± standard deviation. A Student's t-test was performed to assess significance and P<0.05 was considered to indicate a statistically significant difference.

To determine the influence of increased and decreased miR-183 expression in human melanoma, A375 cells were transfected with miR-183 mimic and miR-183 inhibitor sequences, which served as the experimental groups. MiRNA mimic and inhibitor NC sequences were utilized as internal reference groups, using a lentiviral plasmid transfection system. A375 cells that were non-transfected and transfected with blank plasmids were used as external references. MiR-183 expression in each group 12 h following transfection was analyzed using RT-qPCR following total RNA isolation. The results demonstrated that the expression of miR-183 in the miR-183 mimic group was >16-fold higher compared with all control groups, while the miR-183 inhibitor group was 5-fold lower compared with control groups (Fig. 1). These results indicated that the established cell line was stable. MiR-183 levels in the mimic and inhibitor NC groups were similar to those of the blank and NC groups, indicating that transfection alone did not affect miR-183 expression. Additionally, no notable morphological changes were observed among A375 cells in the blank, NC, mimic NC and inhibitor NC groups (data not shown). Therefore, A375 cells in mimic NC and inhibitor NC groups alone were utilized as NCs in future experiments.

To assess the influence of miR-183 expression changes on A375 cell migration, a scratch assay was performed as aforementioned. The results demonstrated that the artificial upregulation of miR-183 expression significantly suppressed A375 cell migration, whereas miR-183 downregulation promoted A375 cell migration when compared with their respective controls (Fig. 2). This indicated that miR-183 inhibits the migration of A375 cells in vitro. To further evaluate the influence of miR-183 expression change on A375 cell migration and invasion, a Transwell assay was performed with each group of cells as aforementioned. The results demonstrated that miR-183 overexpression in the miR-183 mimic group significantly inhibited the migration and invasion of A375 cells in vitro compared with the mimic NC group (Fig. 3A and B). Furthermore, miR-183 knockdown in the inhibitor group promoted A375 cell migration and invasion compared with the inhibitor NC group (Fig. 3C and D). Taken together, this indicates that miR-183 suppresses A375 cell migration and invasion in vitro, and migration and invasion are increased by the downregulation of miR-183.

To determine the inhibitory mechanism of miR-183, changes in Ezrin and MMP-9 protein expression in A375 cells were assessed at 12 h following transfection with an miR-183 mimic or inhibitor. Western blotting revealed that Ezrin and MMP-9 were downregulated and upregulated following miR-183 overexpression and knockdown in vitro, respectively (Fig. 4). Considering the well-established association of Ezrin and MMP-9 with the migratory and invasive activity of melanoma, and that these proteins act as direct targets of miR-183 (45,46,49,53), the results demonstrated that miR-183 inhibits A375 cell migration and invasion potentially via the downregulation of Ezrin and MMP-9 expression.