This study was approved by the Ethics Committee of Subei People's Hospital of Jiangsu province. Written informed consent was provided by all of these patients participated in this research. From May 2014 to February 2016, a total of 21 pairs of melanoma tissues and corresponding adjacent normal tissues were gathered from melanoma patients who suffered a surgical resection at Subei People's Hospital of Jiangsu province. None patients had been treated with chemotherapy, radiotherapy or other treatment prior to surgery. All tissue samples were immediately frozen in liquid nitrogen and stored in −80°C until further use.

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

Total RNA was isolated from tissue samples or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. To detect the levels of miR-675, complementary DNA (cDNA) was synthesized from total RNA using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Quantitative PCR was performed using TaqMan MicroRNA PCR Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI Prism 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). To quantify metadherin (MTDH) mRNA expression level, reverse transcription was carried out with PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd.) was used to detect MTDH mRNA expression. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as normalization for miR-675 and MTDH mRNA expression, respectively. Each experiment was performed in triplicate and repeated three times. The relative levels of miRNA and mRNA were analysed via the 2^−ΔΔCq method (23).

The miR-675 mimics and miRNA negative control mimics (miR-NC) were chemically synthesized by GenePharma (Shanghai, China). MTDH overexpression plasmid (pcDNA3.1-MTDH) and empty pcDNA3.1 plasmid were acquired from GeneCopoeia (Guangzhou, China). Cells were seeded into 6-well plates at a density of 5×105 cells per well. Cell transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as recommended by the manufacturer's instructions. The medium was replaced with fresh DMEM medium containing 10% FBS at 8 h following transfection.

MTT assays were employed to evaluate the proliferative ability of the melanoma cells. Transfected cells were collected at 24 h following transfection, and plated into 96-well plates at a density of 3,000 cells/well. At the indicated time (0, 24, 48, or 72 h incubation), MTT assay was performed according to the manufacturer's instructions. Briefly, 20 µl of MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) reagent was added into each well. After incubation at 37°C with 5% CO₂ for 4 h, the medium was removed followed by addition of 150 µl of DMSO (Sigma-Aldrich; Merck KGaA). Finally, the absorbance was measured using a microplate reader (Omega Bio-Tek, Inc., Norcross, GA, USA) at a wavelength of 490 nm. Experiments were independently repeated in triplicate.

The invasiveness of melanoma cells was determined using Transwell chambers (24-well plate, 8 µm pore; Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Bioscience, San Jose, CA, USA). Transfected cells were collected after 48 h of incubation. 5×10⁴ transfected cells in 200 µl FBS-free DMEM medium were seeded in the upper chambers. The lower chambers were filled with DMEM with 20% FBS as a chemoattractant. Following incubation at 37°C for 24 h, the cells remaining on the upper chambers were removed using cotton swabs. The invasive cells were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet and dried in air. A total of five randomly selected fields were then examined under an inverted microscope (IX73; Olympus Corporation, Tokyo, Japan) at a magnification of 200x. Each assay was performed in triplicate and repeated at least three times.

To predict the potential targets of miR-675, bioinformatics analysis was performed using TargetScan (www.targetscan.org) and miRBase (http://www.mirbase.org/).

MTDH was identified as a candidate target of miR-675 using bioinformatics analysis. A wild-type (Wt) MTDH 3′-UTR containing the binding sequences of miR-675 (pGL3-MTDH-3′-UTR Wt) and a mutant type (Mut) MTDH 3′-UTR lacking the binding sequences of miR-675 (pGL3-MTDH-3′-UTR Mut) were chemically synthesized by GenePharma. Cells were plated into 24-well plates at a density of 1.5×10⁵ cells/well. After incubation overnight, cells were cotransfected with miR-675 mimics or miR-NC, and pGL3-MTDH-3′-UTR Wt or pGL3-MTDH-3′-UTR Mut, using Lipofectamine 2000 in accordance with the manufacturer's instructions. Luciferase activities were measured 48 h after transfection using a Dual-Luciferase^® Reporter Assay System (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. Renilla luciferase activity served as an internal reference.

Total protein was extracted from tissues or cells using cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The concentration of total protein was examined using a bicinchoninic acid (BCA) Protein Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Equal quantity of total protein was separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% skimmed milk in TBS/0.1% Tween (TBST) at room temperature for 1 h and incubated overnight at 4°C with primary antibodies: mouse anti-human monoclonal MTDH (sc-517220; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-human monoclonal GAPDH (sc-66163; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.). Following three washes with TBST, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (sc-2005; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Finally, the protein bands were visualized with enhanced chemiluminescence (ECL) reagents (Bio-Rad Laboratories, Hercules, CA, USA), and analyzed with ImageJ 1.49 (National Institutes of Health, Bethesda, MD). GAPDH served as a loading control.

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

To investigate the roles of miR-675 in the progression of melanoma, RT-qPCR was performed to measure miR-675 expression in 21 paired melanoma tissues and corresponding adjacent normal tissues. As shown in Fig. 1A, miR-675 was significantly downregulated in melanoma tissues compared with that in corresponding adjacent normal tissues (P<0.05). In addition, the analysis of miR-675 expression in four human melanoma cell lines (A375, A2058, HT144 and SK-MEL-28) and the human epidermal melanocytes (HEMs) indicated that expression levels of miR-675 were lower in melanoma cell lines (Fig. 1B; P<0.05). These results suggested that miR-675 may serve important roles in melanoma formation and progression.

miR-675 is lowly expressed in melanoma and thus might serve as a tumour suppressor. Therefore, we determined whether the deregulation of miR-675 in malignant melanoma cells affects biological behaviours of melanoma. A375 and HT144 cells, which exhibited relatively low miR-675 expression, were chosen for subsequent functional assays and transfected with miR-675 mimics or miR-NC. The data from RT-qPCR analysis confirmed that miR-675 was markedly upregulated in A375 and HT144 cells after transfection with miR-675 mimics (Fig. 2A; P<0.05).

An MTT assay was conducted to explore effect of miR-675 overexpression on melanoma cell proliferation. Restoration expression of miR-675 suppressed the proliferation of A375 and HT144 compared with the miR-NC group (Fig. 2B; P<0.05), indicating that miR-675 may serve a suppressive role in the proliferation of melanoma cells. To further characterise the effect of miR-675 on cell invasion ability, Transwell invasion assays were performed. As depicted in Fig. 2C, the upregulation of miR-675 decreased the invasion capacities of A375 and HT144 cells, relative to the miR-NC group (P<0.05). Collectively, these data suggested that miR-675 may play tumour-suppressing roles in melanoma.

To investigate the underlying mechanism responsible for miR-675-mediated tumour-suppressing roles in melanoma, bioinformatics analysis was performed to predict the potential targets of miR-675. Among these potential target genes, MTDH (Fig. 3A) caught our attention because it is upregulated in melanoma and participated in the regulation of biological behaviours of melanoma (24–26). Luciferase reporter assays were used to verify a direct interaction between miR-675 and the 3′-UTR of MTDH. miR-675 mimics or miR-NC was transfected into A375 and HT144 cells, together with pGL3-MTDH-3′-UTR Wt or pGL3-MTDH-3′-UTR Mut. As shown in Fig. 3B, miR-675 overexpression decreased the luciferase activities of the MTDH with a wild-type 3′-UTR vector (P<0.05) but not the mutant vector, implying that miR-675 directly binds the 3′-UTR of MTDH. To further confirm whether miR-675 has regulation effect on MTDH endogenous in melanoma, we detected MTDH mRNA and protein expression in A375 and HT144 cells after transfection with miR-675 mimics or miR-NC. The results revealed that MTDH mRNA (Fig. 3C; P<0.05) and protein (Fig. 3D; P<0.05) expressions were significantly suppressed by miR-675 overexpression in A375 and HT144 cells. Overall, these results suggested that MTDH is a direct target gene of miR-675 in melanoma.

To further explore the relationship between miR-675 and MTDH, we measured MTDH mRNA expression in melanoma tissues and corresponding adjacent normal tissues. The data from RT-qPCR analysis revealed that that levels of MTDH mRNA were significantly upregulated in melanoma tissues compared with those in adjacent normal tissues (Fig. 4A; P<0.05). In addition, Western blotting analysis indicated that MTDH protein was increased in melanoma tissues than adjacent normal tissues (Fig. 4B). Furthermore, Spearman's correlation analysis demonstrated an inverse association between MTDH mRNA and miR-675 expression levels in melanoma tissues (Fig. 4C; r=−0.7251; P=0.0002). These results suggested that increased levels of MTDH in melanoma, at least in part, may be attributed to a downregulation in miR-675.

To further determine whether MTDH is a functional target of miR-675 in melanoma, we performed a rescue experiment involving the transfection of MTDH overexpression plasmids into miR-675-expressing A375 and HT144 cells. As shown in Fig. 5A, the decreased expression of MTDH protein was markedly restored by the transfection of pcDNA3.1-MTDH in A375 and HT144 cells (P<0.05). Functional experiments demonstrated that the upregulation of MTDH could effectively reverse the tumour-suppressing effects on cell proliferation (Fig. 5B; P<0.05) and invasion (Fig. 5C; P<0.05) induced by miR-675 overexpression in A375 and HT144 cells. These results suggested that miR-675 exerted its suppressive effects in melanoma, at least partially, by repressing MTDH.