A total of 49 patients with melanoma were recruited to the present study in the Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from October 2013 to August 2017. Patients diagnosed with other severe diseases and mental health problems were excluded. Patients were diagnosed via imaging examinations and pathological tests. The patients were then divided into 3 tumor stage groups according to tumor thickness. These groups were characterized by the following diagnostic criteria: Stage I, tumor thickness <1.0 mm; stage II, tumor thickness between 1.0 and 4.0 mm; and stage III, tumor thickness >4.0 mm. Patients in stage I included 7 males and 8 females, with an age range of 21 to 68 years and an average age of 43±11.6 years; patients in stage II included 8 males and 9 females, with an age range of 27 to 72 years and an average age of 48±13.1 years; patients in stage III included 8 males and 7 females, with an age range of 24 to 73 years and an average age of 46±11.0 years. No significant differences in age, sex and other basic information were found among different stages. All patients received surgical resection, and tumor tissues and surrounding healthy tissue (within 1 cm around tumor) were collected during the operation. The present study was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital, and all patients provided written informed consent.

Human melanoma cell lines C32 and SK-MEL-28, and the normal skin cell line CCD-1059Sk were all purchased from American Type Culture Collection (Manassas, VA, USA). Cell culture was performed according to manufacturer's protocol in Eagle's minimum Essential medium (EMEM; American Type Culture Collection) containing 10% fetal bovine serum (FBS, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells were harvested during the logarithmic growth phase for subsequent experiments.

Total RNA was extracted from tumor tissues, adjacent healthy tissues and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Tissues were ground in liquid nitrogen before the addition of TRIzol reagent. RNA quality was determined using a NanoDrop^™ 2000 Spectrophotometer (Thermo Fisher Scientific, Inc.), and only RNA samples with a A260/A280 ratio between 1.8 and 2.0 were used to synthesize cDNA via reverse transcription using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.). Reaction conditions for reverse transcription were: 65°C for 5 min, 50°C for 50 min and 85°C for 5 min. A qPCR reaction system was prepared using cDNA and SYBR^®-Green Real-Time PCR Master mix (Thermo Fisher Scientific, Inc.). Following primers were used in the PCR reactions: 5′-TGAGCTCTCAGGAGGGAGGATGG-3′ (forward) and 5′-TTGTCACGTCCACCGGACCTG-3′ (reverse) for H19; 5′-GACCTCTATGCCAACACAGT-3′ (forward) and 5′-AGTACTTGCGCTCAGGAGGA-3′ (reverse) for β-actin. The qPCR thermocycling conditions were as follows: 95°C for 42 sec, followed by 40 cycles of 95°C for 10 sec and 60°C for 35 sec. Data were analyzed using the 2^−ΔΔCq method (12), and the relative expression levels of H19 were normalized to those of the endogenous control β-actin.

H19 siRNA silencing cell lines were constructed using commercial siRNAs. Silencer^™ Select Negative Control No. 1 siRNA (cat. no. 4390843; Thermo Fisher Scientific, Inc.) and H19 siRNA (cat. no. 1299001; Thermo Fisher Scientific, Inc.) were used. Prior to transfection, cells were cultured EMEM containing 10% FBS overnight to reach 80–90% confluence. Lipofectamine 2000^™ reagent (cat. no. 11668-019; Invitrogen, Thermo Fisher Scientific, Inc.) was used to transfect 40 nM siRNA into 5×10⁵ cells by incubation with the cells at 37°C in a CO₂ incubator for 4 h according to the manufacturer's protocol, followed by incubation in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C for 48 h prior to subsequent experimentation.

Cell proliferation ability was measured using a Cell Counting kit-8 (CCK-8; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, 100 µl cell suspension containing 2×10⁴ cells was transferred to each well of a 96-well plate. CCK-8 solution (10 µl) was added into each well for cell culture for 24, 48, 72 and 96 h. Following incubation at 37°C for a further 4 h, the optical density values were measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were seeded into a 24-well plate at 1×10⁵ cells per well. Once cell adhesion was reached (following 48 h), a pipette tip was used to scratch the cells. Cells were washed with PBS and cultured in an incubator (37°C, 5% CO₂) for 24 h to reach ~70–80% confluence. Cell migration was observed under an inverted microscope (Olympus Corporation, Tokyo, Japan), 10 fields of view were randomly selected and images were obtained. ImagePro Plus version 5.0 software (National Institutes of Health, Bethesda, MD, USA) was used to analyze all images.

Transwell cell invasion assay (BD Biosciences, Franklin Lakes, NJ, USA) was performed to measure the cell invasion ability. The upper chamber was pre-coated with Matrigel (cat. no. 356234; EMD Millipore, Billerica, MA, USA) and filled with serum-free RPMI-1640 medium (Thermo Fisher Scientific, Inc.) containing 5×10⁴ cells. RPMI-1640 medium containing 20% fetal calf serum (Sigma-Aldrich, Merck KGaA) was used to fill the lower chamber. Following incubation at 37°C for 24 h, membranes were collected and stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min. A total of 10 fields of vision were randomly selected and stained cells were counted under an optical microscope (Olympus Corporation).

Following protein extraction using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.), the concentration of protein samples was measured using a Bicinchoninic Acid assay. Then, 10% SDS-PAGE gel electrophoresis was performed using 30 µg of protein from each sample, followed by transfer to a polyvinylidene difluoride membrane under 25 V for 1 h. Blocking was performed using 5% skimmed milk at room temperature for 1 h. Following washing with TBST (0.1% Tween-20), membranes were then incubated with the corresponding primary antibodies including rabbit anti-phosphorylated (p)-phosphoinositide 3-kinase (PI3K) antibody (1:2,000; cat. no. ab182651; Abcam, Cambridge, UK), anti-PI3K antibody (1:2,000; cat. no. ab5451; Abcam), anti-p-protein kinase B (AKT) antibody (1:2,000; cat. no. ab18206; Abcam), anti-AKT antibody (1:2,000; cat. no. ab126811; Abcam), anti-inhibitor of nuclear factor κβ (IκBα) antibody (1:1,000; cat. no. ab76429; Abcam), anti-NF-κB p65 antibody (1:1,000; cat. no. ab16502; Abcam), anti-NF-κB p50 antibody (1:1,000; cat. no. ab32360; Abcam) and anti-β-actin primary antibody (1:1,000; cat. no. ab8226; Abcam) overnight at 4°C. Following washing with PBS, membranes were further incubated with anti-rabbit IgG-horseradish peroxidase secondary antibody (1:1,000; cat. no. MBS435036; MyBioSource, San Diego, CA, USA) at room temperature for 1 h. Membranes were then washed with TBST and signals were detected using an enhanced chemiluminescence substrate (Sigma-Aldrich; Merck KGaA) method. Relative expression levels of each protein were normalized to the endogenous control, β-actin, using ImageJ 1.8.0 software (National Institutes of Health).

SPSS software version 19.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Each experiment was performed three times, and results are presented as the mean ± standard deviation. Data between two groups were analyzed by Student's t-test and multiple comparisons were analyzed by one way analysis of variance with a least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to detect the mRNA expression of H19 in the tumor and adjacent healthy tissues of 49 patients with melanoma. As shown in Fig. 1, compared with adjacent healthy tissues, the mRNA expression levels of H19 were significantly increased in the cancerous tissues of 47 of 49 patients (P<0.05), suggesting the potential involvement of H19 in the development of melanoma.

Patients were divided into different stages according to their tumor thickness. As shown in Fig. 2, the expression levels of H19 RNA in tumor tissues were significantly higher in patients with stage II than in patients with stage I (P<0.05). Similarly, the expression levels of H19 RNA in tumor tissues were also significantly higher in patients with stage III compared with patients with stage II (P<0.05). These results suggested that the expression of H19 was greater with increasing stages of melanoma.

As shown in Fig. 3A, compared with the normal skin cell line CCD-1059Sk, expression levels of H19 RNA were significantly increased in the human melanoma cell line C32 and SK-MEL-28 (P<0.05). Following siRNA transfection, the expression levels of H19 RNA were significantly reduced in the C32 and SK-MEL-28 cell lines (P<0.05; Fig. 3A). As shown in Fig. 3B, downregulation of H19 significantly reduced the proliferation ability of C32 and SK-MEL-28 cells (P<0.05). Similarly, downregulation of H19 also significantly reduced the migration and invasion ability of C32 and SK-MEL-28 (P<0.05; Fig. 3C and D). The data suggested that H19 knockdown inhibited the proliferation, migration and invasion of melanoma cells.

It has been well established that the NF-κB signaling pathway may be activated by the PI3K/Akt signaling pathway via the degradation of IκBα. As shown in Fig. 4, no differences in the expression levels of PI3K and Akt were observed between control cells and cells transfected with siRNA. Compared with cells without siRNA transfection, the expression levels of p-PI3K and p-Akt were decreased in cells transfected with siRNA, indicating inactivation of the PI3K/Akt signaling pathway following H19 knockdown. In addition, compared with cells without siRNA transfection, expression levels of the IκBα protein were increased in cells with siRNA transfection, indicating reduced degradation of IκBα following H19 knockdown. These data suggested that H19 knockdown inactivated the PI3K/Akt signaling pathway, which in turn inhibited the activation of the NF-κB signaling pathway.

The expression of NF-κB p65 and p50 may be regulated by certain lncRNAs. Therefore, the effect of H19 knockdown on the expression levels of NF-κB p65 and p50 were investigated. As shown in Fig. 5, compared with the cells without siRNA transfection, expression levels of NF-κB p65 and p50 were decreased in cells following siRNA transfection. These data suggested that H19 knockdown inhibited the NF-κB signaling pathway by downregulating the expression levels of NF-κB p65 and p50.