A total of 23 pairs of melanoma tissues and adjacent non-tumour tissues were collected from patients (male, 14; female, 9; age, 36–69 years; mean age, 52 years) at the Tangshan City Workers' Hospital between January 2013 and October 2015. All patients involved in this study were not treated with radiation therapy or chemotherapy prior to the surgical resection. These resected tissues were immediately frozen in liquid nitrogen and then stored at −80°C until use. The present study was approved by the Ethics Committee of the Institute of Tangshan City Workers' Hospital. In addition, informed consents were obtained from all subjects prior to the study.

Four human melanoma cell lines, namely SK-MEL-28, A375, HT144 and A2058, were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific), 100 mg/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific). Human epidermal melanocytes (HEMs) were purchased from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) and cultured in melanocyte medium (ScienCell Research Laboratories) according to the manufacturer's protocol. All cells were grown in a humidified incubator at 37°C with 5% CO₂.

miR-326 mimics, miRNA negative control mimics (miR-NC), a small interfering RNA (siRNA) targeting KRAS (KRAS siRNA) and a negative control siRNA (NC siRNA) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). pcDNA3.1-KRAS plasmid and empty pcDNA3.1 plasmid were synthesised by GeneCopoeia (Guangzhou, China). The cells were transfected with miRNAs, siRNAs or plasmids using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions. The cell culture medium was replaced with fresh DMEM supplemented with 10% FBS at 6 h post-transfection.

Total RNA samples were isolated from the tissues or the cells using TRIzol (Invitrogen; Thermo Fisher Scientific), according to the manufacturer's instructions. For the miR-326 detection, total RNA was reverse transcribed into cDNA using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA) and qPCR was carried out with a TaqMan MicroRNA PCR kit (Applied Biosystems) on an ABI Prism 7500 Sequence Detection system (Applied Biosystems). To quantify KRAS mRNA, cDNA was synthesised using PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China), followed by qPCR with SYBR Premix Ex Taq™ (Takara Biotechnology). U6 and GAPDH were used as internal reference for miR-326 and KRAS, respectively. The relative levels of miRNA and mRNA were analysed via the 2^−ΔΔCt method (26). The primers used in the present study were as follows: miR-326 forward, 5′-GGCGCCCAGAUAAUGCG-3′ and reverse, 5′-CGTGCAGGGTCCGAGGTC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′; KRAS forward, 5′-GACTCTGAAGATGTACCTATGGTCCTA-3′ and reverse, 5′-CATCATCAACACCCTGTCTTGTC-3′; GAPDH forward, 5′-ATGGGTCAGAAGGATTCCTATGTG-3′ and reverse, 5′-CTTCATGAGGTAGTCAGTCAGGTC-3′.

A CCK8 assay was used to assess melanoma cell proliferation. A total of 3×10³ cells/well were seeded into 96-well plates. After being cultured overnight at 37°C, the cells were transfected with miRNAs, siRNAs or plasmids. The examination time-points were set at 0, 24, 48 and 72 h after transfection. In brief, 10 µl of CCK8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well and the cells were incubated at 37°C with 5% CO₂ for 2 h. A microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) was used to measure the optical density (OD) values at a wavelength of 450 nm. Each experimental group contained five replicate wells and this assay was repeated three times.

The Transwell chambers coated with Matrigel (BD Biosciences, San Jose, CA, USA) on the upper surface of a polycarbonic membrane were used to perform cell invasion assays. A total of 1×10⁵ transfected cells in FBS-free DMEM were placed in the upper chambers. The bottom chambers were filled with 500 µl of DMEM containing 20% FBS serving as a chemoattractant. After 24 h of incubation, the cells remaining on the upper surface of the membrane were removed using cotton swabs. The invasive cells were fixed in 90% alcohol, stained with 0.5% crystal violet, dried at 80°C for 30 min and photographed. The cell number was determined in five fields randomly selected under an inverted microscope (×200 magnification, IX73; Olympus Corporation, Tokyo, Japan). The experiments were performed in triplicates and repeated three times.

At 48 h after transfection, the cells were collected via trypsinization, washed with ice-cold phosphate-buffered saline (PBS) and fixed in ice-cold 80% ethanol in PBS. Fluorescein isothiocyanate (FITC) Annexin V-apoptosis detection kit (BD Biosciences) was used to determine cell apoptosis rate according to the manufacturer's instructions. In brief, the fixed cells were treated with FITC Annexin V and propidium iodide (PI) for 20 min in the dark at room temperature. The cell apoptosis rate was assessed using FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) within 1 h of staining.

Bioinformatic analysis was performed to predict the putative targets of miR-326 using TargetScan (www.targetscan.org) and miRBase (http://www.mirbase.org). KRAS was indicated as a potential target of miR-326.

For the luciferase activity assay, a wild-type and mutant 3′-UTR segment of KRAS cloned into a pmirGLO plasmid (pmirGLO-KRAS-3′-UTR Wt and pmirGLO-KRAS-3′-UTR Mut) was synthesised and confirmed by Shanghai GenePharma. The cells were seeded into 24-well plates at a density of 60–70% confluency and co-transfected with miR-326 mimics or miR-NC and pmirGLO-KRAS-3′-UTR Wt or pmirGLO-KRAS-3′-UTR Mut using Lipofectamine 2000. After incubation at 37°C for 48 h, the cells were harvested, and luciferase activity was assessed using the Dual-Luciferase Reporter assay system (Promega, Manheim, Germany) according to the manufacturer's protocol. Renilla luciferase activity served as an internal reference. The experiments were performed ≥3 times independently.

The protein was extracted from tissues or cells using radioimmunoprecipitation assay buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) supplemented with a freshly added protease inhibitor cocktail (Roche, Manheim, Germany). After centrifuging at 16,000 × g at 4°C for 10 min, the protein concentration was detected using the Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific). Equal amounts of protein were dissolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in Tris-buffered saline, 0.1% Tween (TBST) at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used in this assay included mouse anti-human monoclonal KRAS (sc-30; 1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human monoclonal p-AKT (sc-81433; 1:1,000 dilution; Santa Cruz Biotechnology), mouse anti-human monoclonal AKT (sc-56878; 1:1,000 dilution; Santa Cruz Biotechnology), mouse anti-human monoclonal p-ERK (sc-81492; 1:1,000 dilution; Santa Cruz Biotechnology), mouse anti-human monoclonal ERK (sc-514302; 1:1,000 dilution; Santa Cruz Biotechnology), and mouse anti-human monoclonal GAPDH antibody (sc-32233; 1:1,000 dilution; Santa Cruz Biotechnology). The membranes were then washed three times with TBST for 5 min each time and further probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. The signals were developed using an enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ, USA). The signal intensity was determined using the FluorChem imaging system (Alpha-InnoTec GmbH, Kasendorf, Germany). GAPDH was used as a loading control.

The data were expressed as the mean ± standard deviation. All statistical analyses were performed via the Student's t-test or one-way ANOVA followed by Student-Newman-Keuls post hoc test, with SPSS 18.0 software (SPSS, Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To explore the potential roles of miR-326 in melanoma, we firstly detected the expression levels of miR-326 in 23 pairs of melanoma tissues and adjacent non-tumour tissues. The RT-qPCR data revealed that miR-326 was weakly expressed in melanoma tissues compared with that in adjacent non-tumour tissues (Fig. 1A, P<0.05). Subsequently, the expression levels of miR-326 were determined in four melanoma cell lines, namely SK-MEL-28, A375, HT144 and A2058 as well as HEM, using RT-qPCR. The results revealed that miR-326 was significantly downregulated in melanoma cell lines compared with that in HEM (Fig. 1B, P<0.05). These findings indicated that miR-326 may contribute to melanoma development.

To assess the regulatory roles of miR-326 in melanoma, SK-MEL-28 and A375 melanoma cells, which exhibited a relatively weaker miR-326 expression, were transfected with miR-326 mimics or miR-NC. The RT-qPCR data indicated that the miR-326 expression was substantially upregulated in the SK-MEL-28 and A375 cells after transfection with miR-326 mimics, compared with cells transfected with miR-NC (Fig. 2A, P<0.05). The CCK8 assay was performed to evaluate the effect of miR-326 overexpression on melanoma cell proliferation. As illustrated in Fig. 2B, the restoration of the expression of miR-326 significantly suppressed the proliferation of the SK-MEL-28 and the A375 cells compared with the miR-NC group (Fig. 2B, P<0.05). To determine the role of miR-326 in the invasion capability of melanoma cells, cell invasion assays were conducted in the SK-MEL-28 and A375 cells transfected with miR-326 mimics or miR-NC. The results revealed that ectopic expression of miR-326 decreased the invasive capacities of the SK-MEL-28 and A375 cells (Fig. 2C, P<0.05). To examine the effect of miR-326 on the apoptosis of melanoma cells, flow cytometric analysis was performed. Our data demonstrated that miR-326 overexpression led to an increased rate of apoptosis in the SK-MEL-28 and A375 cells (Fig. 2D, P<0.05). Overall, these findings indicated that miR-326 exerts a suppressive role in melanoma progression.

To investigate the underlying mechanism responsible for the miR-326-mediated tumour-suppressing roles in melanoma, the potential binding sites of miR-326 were determined using bioinformatic analysis. Of these target candidate genes, KRAS (Fig. 3A) attracted our attention immediately since it has been implicated in tumourigenesis and progression (27–29). To confirm this hypothesis, luciferase reporter assays were performed in SK-MEL-28 and A375 cells transfected with miR-326 mimics or miR-NC along with pmirGLO-KRAS-3′-UTR Wt or pmirGLO-KRAS-3′-UTR Mut. As displayed in Fig. 3B, miR-326 significantly decreased the luciferase activities of the 3′-UTR Wt (P<0.05), but not the 3′-UTR Mut of KRAS in the SK-MEL-28 and A375 cells. To further confirm whether KRAS is a direct target of miR-326, the effects of miR-326 overexpression on endogenous KRAS expression in melanoma cells were examined by RT-qPCR and western blot analysis. The results revealed that restoration of the expression of miR-326 suppressed the KRAS expression in the SK-MEL-28 and A375 cells at both the mRNA (Fig. 3C, P<0.05) and protein (Fig. 3D, P<0.05) levels. These results indicated that KRAS is a novel target of miR-326 in melanoma.

We further determined the expression levels of KRAS in 23 pairs of melanoma tissues and adjacent non-tumour tissues. The results revealed that KRAS mRNA was highly expressed in melanoma tissues compared with that in adjacent non-tumour tissues (Fig. 4A, P<0.05). In addition, a negative correlation between the miR-326 level and the KRAS level was observed in melanoma tissues (Fig. 4B, r=−0.6589, P<0.001), further indicating that the KRAS overexpression in melanoma is a result of the miR-326 underexpression. In addition, western blot analysis also revealed that the KRAS expression was upregulated in melanoma tissues compared with that in adjacent non-tumour tissues (Fig. 4C).

Considering that KRAS is a direct target of miR-326, we hypothesised that the tumour-suppressing roles of miR-326 in melanoma cells could be achieved by KRAS downregulation. To examine this hypothesis, we knocked down KRAS via RNA interference techniques to evaluate the biochemical roles of KRAS in melanoma. Western blot analysis confirmed that KRAS protein was significantly downregulated in the SK-MEL-28 and A375 cells after transfection with KRAS siRNA (Fig. 5A, P<0.05). Functional assays revealed that the downregulation of KRAS inhibited cell proliferation (Fig. 5B, P<0.05) and invasion (Fig. 5C, P<0.05) and promoted apoptosis (Fig. 5D, P<0.05) in the SK-MEL-28 and A375 cells. These results indicated that KRAS mediates the biological functions of miR-326 in melanoma.

To determine whether miR-326 induces antitumour effects in melanoma cells by targeting KRAS, rescue experiments were performed. The SK-MEL-28 and A375 cells were transfected with miR-326 mimics with or without pcDNA3.1-KRAS. Western blot analysis revealed that decreased KRAS expression was markedly restored by transfection of pcDNA3.1-KRAS (Fig. 6A, P<0.05). Expectedly, the KRAS overexpression markedly reversed the miR-326-induced tumour suppressive effects in the SK-MEL-28 and A375 cells (Fig. 6B-D, P<0.05). These results demonstrated that miR-326 served as a tumour suppressor in melanoma, at least in part through the KRAS suppression.

Previous studies reported that KRAS activated the AKT and ERK signalling pathways (30,31). Subsequently, we examined whether the miR-326 upregulation could inhibit the AKT and ERK signalling pathways. As illustrated in Fig. 7, the miR-326 upregulation reduced the p-AKT (P<0.05) and p-ERK (P<0.05) expression in the SK-MEL-28 and A375 cells, however it had no significant effects on the AKT and ERK expression. These results indicated that miR-326 regulated the AKT and ERK signalling pathways in melanoma.