A total of 16 melanoma tissues and normal skin samples were collected from patients recruited from the Department of Plastic Surgery, Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) between February 2015 and June 2016. The mean age of the recruited patients (10 male and 6 female) were 56.46 years (standard deviation, 2.63 years). The fresh tissues were frozen in liquid nitrogen (−196°C) to protect the protein or RNA from degradation. The use of human tissues was approved by the Ethics Committee of the Central Hospital of Wuhan and all patients provided written, informed consent.

The human melanoma cell lines A375, WM115 and WM75 were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), and were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), penicillin (25 U/ml), streptomycin (25 g/ml) and 1% L-glutamine. Normal human melanocytes were purchased from Lifeline Cell Technology, LLC (Frederick, MD, USA; cat. no. FC-0030) and grown in LL-0027 medium (Lifeline Cell Technology, LLC). All cell lines were cultured in a 5% CO₂ and 37°C incubator. For the upregulation of miR-373 expression, a synthesized miR-373 mimic (5′-GAAGUGCUUCGAUUUUGGGGUGU-3) and a miR-NC (5′-UUCUCCGAACGUGUCACGUTT-3′ (both 10 nM; Shanghai GenePharma Co., Ltd., Shanghai, China) was transfected into A375 or WM115 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The medium containing the transfection reagents was removed 6 h after transfection and 48 h later the cells were harvested for subsequent assays. The transfection efficiency and changes in miR-373 expression were determined by RT-qPCR. For the downregulation of SIK1 expression, short hairpin (sh)RNA against SIK1 (sh-SIK1; Shanghai GenePharma Co., Ltd.) was stably transfected in A375 or WM115 cells using Lipofectamine 2000, according to the manufacturer's protocol. The sh-SIK1 sequence was 5-UGAACAAGAUCAAAGGGUU-3 and negative control shRNA (sh-NC) sequence was 5-AATTCTCCGAACGTGTCACGT-3 (Hanyin Biotechnology, Shanghai, China). The transfection efficiency and changes in SIK1 expression were determined by western blotting. The medium containing the transfection reagents was removed 6 h after transfection and 48 h later the cells were harvested for the following assays.

To determine whether overexpression of miR-373 promoted melanoma through targeting of the 3′-UTR of its downstream gene, three different algorithms [TargetScan (http://www.targetscan.org/vert_61/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) and miRDB (http://mirdb.org/miRDB/)] were used to select the potential target genes of miR-373.

A375 or WM115 cells were cultured in a 25 cm² culture flask to 80–90% confluence in DMEM supplemented with 10% FBS at 37°C, collected by digestion with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) and centrifuged at 4°C at 1,409 × g for 5 min, and then seeded into 6-well plates at 2.5×10⁵ cells/well. miR-373 mimic, miR-NC, sh-SIK1 and sh-NC were transfected into A375 or WM115 cells as described above. Streaks were created in the monolayer with a pipette tip. Progression of migration was observed and photographed 24 h after wounding. The floating cells were removed by washing with PBS, and the width of scratch was observed at 0 and 24 h using an inverted microscope (magnification, ×50). Each experiment was repeated three times.

The migration capability of A375 or WM115 cells was determined using a Transwell assay. The two cell lines were harvested and seeded with serum-free DMEM into the upper chambers at 5×10⁴ cells/well. The bottom chambers contained DMEM with 10% FBS. The Transwells were incubated for 24 h at 37°C. Following incubation, the migrated cells attached to the lower surface of the membrane were fixed by 4% paraformaldehyde for 30 min at room temperature and stained with 1% toluidine blue for 15 min at room temperature. Cell numbers were counted in five randomly chosen fields using an inverted microscope (magnification, ×100) per membrane and counts were repeated three times.

Total RNA from tissues or cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized using a PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. qPCR was performed with a KAPA SYBR FAST qPCR kit (Kapa Biosystems, Inc., Wilmington, MA, USA) using a 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The initial denaturation step was for 10 sec at 95°C, followed by 40 cycles of denaturation for 5 sec at 95°C, and annealing and extension for 20 sec at 60°C. The expression levels of miRNA were normalized to endogenous small nuclear RNA U6. The 2^−ΔΔCq method was used to analyze the expression level relative to the endogenous control (20). The primers used were as follows: miR-373 forward, 5′-ATTTTGGTTAATACGGTGAAATTTC-3′ and reverse, 5′-CTATCGCCCAAACTAAAATACGAT-3′; SIK1 forward, 5-TGGACGTCTGGAGCCTCGGT-3′ and reverse, 5′-CTCGCGTTTTTCCTTAGCTG-3′; U6, forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-ACGCTTCACGAATTTGCGT-3′.

Protein extraction and western blotting analysis were conducted as described previously (21). The cells were incubated with the primary antibodies, including anti-SIK1 (cat. no. ab64428; 1:1,000; Abcam, Cambridge, MA, USA) or anti-GAPDH (cat. no. sc-25778; 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Then the membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc.).

For the luciferase reporter assay, A375 cells were seeded on 24-well plates and co-transfected using Lipofectamine 2000 with 100 ng/well firefly luciferase UTR-reporter vector, 2 ng/well Renilla pRLCMV vector (internal control; all Promega Corporation, Madison, WI, USA) and 20 ng/well of miR-373 mimics or miR-NC (Applied Biosystems; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. In brief, luciferase reporters were constructed through cloning of the 3′-UTR of SIK1 wild-type (wt) as well as SIK1 mutant-type (mut) via a deletion at position 1,905–1,911. The 3UTR of SIK1 mRNA containing the wild-type or mutant miR-373 recognition sequences were PCR-amplified and subcloned into the SacI and SalI sites of the pmirGLO vector (Promega Corporation). A375 cells transfected with miR-373 or miR-NC and cultured in 48-well plates were co-transfected with 1.5 µg of firefly luciferase reporter and 0.35 ng Renilla luciferase reporter with Lipofectamine 2000 regent. After 24 h the relative luciferase activity was assessed with the Dual-Luciferase Assay Reporter system (cat. no. E1910; Promega Corporation) and normalized to Renilla luciferase activity.

Data are presented as the mean ± standard deviation from at least three independent experiments. All data were analyzed using GraphPad 6.0 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). Differences between two groups were analyzed using two-independent-sample Student's t-test or non-parametric Mann-Whitney U test. Differences between multiple groups were analyzed using one-way analysis of variance with Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of miR-373 in melanoma, RT-qPCR was performed. The results indicated that the mRNA level of miR-373 was significantly upregulated in melanoma tissues compared with nevus (Fig. 1A). Next, the expression of miR-373 was evaluated in a panel of normal melanocytes and human melanoma cell lines. The results indicated that miR-373 was significantly upregulated in A375, WM115 and WM75 melanoma cell lines compared with normal melanocytes (Fig. 1B). The results indicated that the expression of miR-373 is increased in melanoma tissues and cell lines.

To explore the functional role of miR-373 in melanoma progression, melanoma cells [A375 (relatively highest level of miR-373 among the three cell groups from Fig. 1B) and WM115 (relatively lowest level of miR-373 among the three cell groups from Fig. 1B)] were stably transfected with miR-373 mimics. It was confirmed that the expression of miR-373 was upregulated in A375 and WM115 cells following miR-373 mimic transfection compared with cells transfected with miR-NC (Fig. 2A). As indicated in Fig. 2B and C, a wound healing assay demonstrated that transfection with miR-373 mimic significantly enhanced melanoma cell migration in A375 and WM115 cells compared with transfection with miR-NC. The effects of miR-373 on melanoma cell migration were evaluated further using a Transwell assay (Fig. 2D and E). A375 and WM115 cells transfected with miR-373 mimic were observed to have a significantly higher migration capability compared with cells transfected with miR-NC. These results suggested that miR-373 elevates melanoma cell migration.

Among the potential candidate targets of miR-373 was SIK1 (Fig. 3A). Fig. 3B indicates that the seed sequence of miR-373 is complementary to the 3′-UTR of SIK1 and is highly conserved among 18 different species. Dual-luciferase reporter assays illustrated that the transient transfection of A375 cells with SIK1 wt 3′-UTR significantly reduced reporter expression when compared with the control vector (Fig. 3C and D). However, the firefly luciferase reporter activity of the SIK1 mut 3′-UTR was not notably affected by miR-373-transfection (Fig. 3D). This result indicated that the 3′-UTR of SIK1 was targeted by miR-373. Furthermore, western blot analysis results demonstrated that overexpression of miR-373 inhibited SIK1 protein level in A375 and WM115 cells (Fig. 3E). These results supported the conclusion that SIK1 is the target gene of miR-373 in melanoma.

The functional role of SIK1 in melanoma was evaluated. The effect of sh-SIK1 and sh-NC on SIK1 expression in A375 and WM115 cell lines was observed via western blot analysis. It was confirmed that transfection with sh-SIK1 resulted in decreased SIK1 expression compared with sh-NC in the two cell lines (Fig. 4A). A375 cells and WM115 cells transfected with sh-SIK1 exhibited significantly increased cell migration compared with sh-NC Transfected cells, as demonstrated by wound healing and Transwell assays (Fig. 4B-E). These results indicated that downregulation of SIK1 promotes melanoma cell progression.