Human malignant melanoma tissues and melanocytic nevus tissues (surgically resected and later histopathologically diagnosed as benign) were obtained from patients with melanoma during surgery, and diagnosed by pathological validation. A total of 10 patients with nevus and melanoma tissues examined by pathology and without other skin diseases from the Changhai Hospital (Shanghai, China) were included in the study, including 7 male and 3 female patients with a mean age of 48 (age range, 36–65). Ten nevus and matched melanoma tissue samples were obtained between September 2005 and September 2008. All samples were snap-frozen in liquid nitrogen until examination. All human samples were collected with the written informed consent of the patients, and use of human tissues was approved by the Institutional Research Ethics Committee of the Second Military Medical University (Shanghai, China).

The human malignant melanoma A375 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (PAA; GE Healthcare, Chicago, IL, USA) with 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), under 37.5°C and 5% CO₂. Cells were transfected with small interfering (si)RNAs using INTERFERin^® reagent (Polyplus-transfection SA, Illkirch, France) according to the manufacturer's protocol, and transfected with MSG1 expressing plasmids (constructed using a pcDNA 3.1 vector) (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using JetPEI^® reagent (Polyplus-transfection SA) following the manufacturer's protocol, and confirmed by western blot analysis, in order to induce MSG1 overexpression. The siRNA target sequences for human MSG1 gene were 5′-UAGCAGCACAUCAGUCGAAUA-3′ (sense) and 5′-CCCAAUAUUGUCAAUUAUUUA-3′ (antisense), and the negative control siRNA sequences were 5′-UCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). siRNA duplexes were transfected at a final concentration of 10 nM.

The Affymetrix GeneChip Human Genome U133 Plus 2.0 array assay was performed by Shanghai Bohao Industrial Co., Ltd., Shanghai, China. In brief, 5 µg total RNA samples from melanoma tissues and paired nevus tissues were reverse transcribed into cDNA for use in the microarray, as previously described (17). Hybridization was performed overnight using a micro-circulation pump (Atactic Technologies, Inc., Houston, TX, USA), and images were collected and quantified (17). The differentially detected signals were gathered and presented.

Total RNA, sourced from patient tissues, was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RT-qPCR analysis was performed using a SYBR RT-PCR kit (Takara Bio, Inc., Otsu, Japan) and LightCycler (Roche Diagnostics, Basel, Switzerland). The primer sequences for MSG1 were 5′-GGCGGCACCACCATGTACCCT-3′ (sense) and 5′-AGGGGCCGGACTCGTCATACT-3′ (antisense); the primer sequences for Bcl-2 were 5′-GGTGGGGTCATGTGTGTGG-3′ (sense) and 5′-CGGTTCAGGTACTCAGTCATCC-3′ (antisense); and the internal control b-actin sequences were 5′-GGCGGCACCACCATGTACCCT-3′ (sense) and 5′-AGGGGCCGGACTCGTCATACT-3′ (antisense). The PCR cycle conditions were 95°C 15 sec, 55°C 30 sec, 72°C 30 sec for 45 cycles, and three independent experimental repeats were performed. The relative expression level of gene mRNAs was normalized to that of internal control β-actin by using 2^−ΔΔCq cycle threshold method (18).

The cell viability of transfected A375 cells was examined by the MTT method. Briefly, cells (10,000) were seeded into 96-well plates, cultured in DMEM with 10% FBS, and transfected as described. Control cells were transfected with empty pcDNA 3.1 vectors. At the indicated time points (0, 48 and 96 h), cell culture medium was replaced by fresh medium containing 0.5 mg/ml MTT. Cells were then incubated at 37°C for 2 h, and the MTT-containing medium was then replaced by 0.1 ml of dimethyl sulfoxide to dissolve the formazan (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The absorbance in each well was detected at 570 nm.

Melanoma cells were transfected for 48 h and cell culture medium was subsequently replaced by serum-free medium (DMEM; PAA; GE Healthcare). At the indicated time points (0 and 48 h), cells were harvested and apoptosis was detected using a Calbiochem^® Annexin V-FITC Apoptosis Detection kit (Merck KGaA) according to the manufacturer's protocol, and a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The Annexin V-positive cells were regarded as apoptotic.

MSG1 expression in melanoma tissues was examined by IHC. In brief, tissues were fixed with formalin and embedded in paraffin, and then sectioned to make tissue sections (4 µm thick), which were deparaffinized in xylene for 20 min and rehydrated in graded ethanol (95, 85, 75 and 50% for 5 min each). Endogenous peroxidase activity was blocked by a 30 min incubation in 3% H₂O₂ in PBS, and antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) by heating to boil for 5 min. The anti-MSG1 primary antibody (cat no. ab87978; Abcam, Cambridge, UK) was diluted 1:500 and incubated at 4°C overnight. The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (cat no. ab97040; Abcam, Cambridge, UK) in 1:1,000 dilution for use and in incubation at 4°C for 2 h. Immunostaining was visualized using a 3,3′-diaminobenzidine staining kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and analyzed using HistoFAXS system and mean DAB staining intensity was calculated using Histoquest software (both from TissueGnostics, Vienna, Austria), and the images are presented in Fig. 1C.

Cells were lysed using Passive Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Protein concentrations were measured using the BCA Protein Assay kit (Takara Bio., Inc.) and equal amounts of extracts (30 µg) were subjected to SDS-PAGE (10% gel), transferred onto a polyvinylidene fluoride membrane, and then blotted. The MSG1 antibody (cat no. ab87978) was purchased from Abcam. B-cell lymphoma 2 (Bcl-2; cat no. CST 2872) and β-actin (cat no. CST 3700) antibodies, all at a 1:1,000 dilution, and horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signaling Technology, Inc. Antibody incubation was performed for 3 h at 4°C. At least 3 replicates were performed. Blocking was performed using tris-buffered saline with Tween-20, with 5% bovine serum albumin for 1 h at 20°C. Imaging was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.), and densitometric analysis was performed using Labworks Image Acquisition and Analysis Software (UVP, Upland, CA, USA).

Results are presented as the mean ± standard deviation. Statistical analyses were performed using a Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the gene expression profile in malignant melanoma, a cDNA microarray was performed in the melanoma and paired nevus tissues, and the differential gene expression is presented in Fig. 1A. Among the differentially expressed genes, it was identified that the melanocyte differentiation-associated gene MSG1 was significantly upregulated in melanoma tissue compared with nevus tissue (P<0.01; Fig. 1B). Additionally, the upregulation of MSG1 was confirmed by IHC in the melanoma tissues compared with nevus tissues (Fig. 1C). The results indicated that MSG1 is upregulated in melanoma, and may potentially participate in melanoma carcinogenesis and progression.

As MSG1 was upregulated in melanoma, the roles of MSG1 in melanoma development were further examined. The cell viability of melanoma A375 cells was examined in control-transfected or MSG1-overexpressing cells, revealing that MSG1 overexpression could significantly increase cell viability in A375 cells (P<0.01; Fig. 2A and B). In addition, this result was confirmed by the knockdown of MSG1 expression. Transfection of the A375 cells with an MSG1-specific siRNA significantly decreased the cell viability compared with the control-siRNA-transfected cells (P<0.01; Fig. 2C and D). Thus, it was concluded that overexpression of MSG1 in melanoma may increase cell viability.

In order to determine the mechanism of MSG1-promoted cell viability, the effect of MSG1 on cell apoptosis were additionally examined in melanoma cells. As demonstrated in Fig. 3A, MSG1 overexpression significantly inhibited cell apoptosis induced by serum deprivation (P<0.01), whereas knockdown of MSG1 expression significantly promoted the serum deprivation-induced cell apoptosis (P<0.01; Fig. 3B). Therefore, MSG1 may inhibit cell apoptosis of melanoma cells, thus enhancing cell viability and promoting melanoma progression.

The molecular mechanisms responsible for the inhibition of apoptosis mediated by MSG1 overexpression were explored. The expression of apoptosis-associated intracellular proteins in MSG1-overexpressing A375 cells were screened, and it was identified that the expression of the anti-apoptotic protein Bcl-2 was significantly increased by MSG1 overexpression at the mRNA (P<0.01; Fig. 4A) and protein levels (Fig. 4B). Furthermore, the knockdown of MSG1 expression significantly inhibited the expression of Bcl-2 in A375 cells (P<0.01; Fig. 4C and D). Taken together, the results indicate that MSG1 may enhance the expression of anti-apoptotic Bcl-2, thus inhibiting cell apoptosis and promoting melanoma progression.