The melanoma cell lines A375, C8161, SK-Mel-5 and Mel-Juso were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). All cultures were maintained in a 5% CO₂ incubator at 37°C. After detecting the Bmi-1 levels in the aforementioned cell lines, the optimal cells were selected for further study.

The siRNA target sequence for the Bmi-1 gene (NM_005180) was designed and the si-Bmi-1 constructs were inserted into the GV248 green fluorescent protein-expressing lentiviral vector (GeneChem, Shanghai, China). After a series of programmes to transfect, concentrate and purify, Bmi-1 shRNA lentivirus and empty lentivirus were obtained with a range of titer yields: 1–1.5×10⁹ TU/ml and transfected into A375 cells (MOI=20). The expression of Bmi-1 in A375 cells after transfection was confirmed by real-time PCR and western blot analysis.

Total RNA from A375, Bmi-1-knockdown (A375-Bmi-1-shRNA) and pMSCV vector control cells (A375-vector) were extracted using the RNeasy Midi kit (Qiagen, Inc., Valencia, CA, USA) and reverse transcribed to cDNA with a PrimeScript RT reagent kit (Takara, Tokyo, Japan). Real-time PCR was performed using SYBR Premix Ex Taq (Takara) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Invitrogen). The cycling conditions were as follows: 95°C for 30 sec, 95°C for 10 sec and 60°C for 30 sec. The following are the gene-specific primer pairs: Bmi-1, 5′-CTGGTTGCCCATTGACAGCG-3′ and 5′-AAATCCCGGAAAGAGCAGCC-3′; E-cadherin, 5′-CGGTGGTCAAAGAGCCCTTA-3′ and 5′-TGAGGGTTGGTGCAACGTCGTTA-3′; α-catenin, 5′-GGCAGCCAAAAGACAACAGG-3′ and 5′-GGCCTTATAGGCTGCGACAT-3′; vimentin, 5′-CAGGCAAAGCAGGAGTCCAC-3′ and 5′-GCAGCTTCAACGGCAAAGTTC-3′; N-cadherin, 5′-CGAATGGATGAAAGACCCATCC-3′ and 5′-GCCACTGCCTTCATAGTCAAACACT-3′; and β-actin, 5′-TGGCACCCAGCACAATGAA-3′ and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The gene expression levels were calculated using the 2^−ΔΔCt method.

Total protein was extracted from the aforementioned cultured cells and quantified. Equal protein amounts were analyzed using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with a non-specific antibody binding with non-fat dried milk for 2 h at room temperature, the membranes were incubated overnight with primary antibodies at 4°C and then incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The primary antibodies used were: anti-Bmi-1 (Abcam, Cambridge, UK), anti-E-cadherin, anti-α-catenin (both from ProteinTech, Chicago, IL, USA), anti-vimentin, anti-N-cadherin (both from Zhongshan Co., Beijing, China), anti-PTEN (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Akt, anti-phospho-Akt (both from Cell Signaling Technology, Inc., Beverly, MA, USA), anti-NF-κB (ProteinTech), anti-phospho-NF-κB (Cell Signaling Technology, Inc.), anti-MMP-2 (ProteinTech) and anti-β-actin (Santa Cruz Biotechnology, Inc.).

The cells were fixed in 4% paraformaldehyde, permeabilized in a solution of 0.3% Triton X-100 and 5% bovine serum albumin (BSA) for 30 min at room temperature and incubated with antibodies against E-cadherin, α-catenin, vimentin or N-cadherin overnight at 4°C. Subsequently the cells were incubated with the appropriate Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 37°C for 1 h. The nuclei were counterstained with DAPI and imaged with a confocal laser-scanning microscope (Nikon, Tokyo, Japan).

After dewaxing and rehydration, the sections were subjected to antigen retrieval by boiling in 10 mM citrate buffer. After incubation with 3% hydrogen peroxide for 20 min to block the endogenous peroxidase activity, the sections were immersed in 5% bovine serum for 30 min to minimize antibody non-specificity. Next, the sections were incubated with antibodies (anti-E-cadherin, anti-α-catenin, anti-vimentin and anti-N-cadherin) overnight at 4°C and then washed. The sections were then treated with secondary antibodies (horseradish peroxidase-labeled goat anti-mouse/rabbit; Zhongshan Co.) for 30 min at 37°C. Following development by diaminobenzidine (Zhongshan Co.), the sections were counterstained with hematoxylin and evaluated using microscopy.

The Transwell polycarbonate membranes containing 8-µm pores were coated with Matrigel (BD Biosciences, San Jose, CA, USA). Cells were resuspended in serum-free DMEM and seeded into the upper wells, and DMEM supplemented with 15% bovine serum was placed into the lower chamber (6). The cells that migrated through the membranes were observed with crystal violet staining after incubation for 24 h at 37°C.

Six-week-old female BALB/c-nude mice were obtained. Animals were housed in pathogen-free conditions with filtered air, autoclaved food and water was available. A375-vector, A375-Bmi-1-shRNA and 4×10⁶ cells in serum-free DMEM were injected subcutaneously into mice on their right flanks (six mice/group). Tumor xenografts were measured once/week with calipers. Mice were observed with live small-animal imaging technology at the end of the fifth week. Tumor xenografts were harvested for histological analysis. The volume was calculated as: ab²/2 (a and b represent the length and width of a tumor, respectively) and the tumor weights were measured. These data were statistically compared by a two-sample t-test, with a significance level of P<0.05. The histology and immunohistochemistry of the tumors were observed under a microscope.

To investigate the impact of Bmi-1 on EMT, we compared the expression level of Bmi-1 in several melanoma cell lines, and the A375 cell line was selected for further study (Fig. 1A). We silenced endogenous Bmi-1 in the A375 cells using specific shRNAs and knockdown effects were detected by real-time PCR and western blotting. All three shRNAs specifically knocked down the mRNA levels of Bmi-1. Due to the best efficacy of shRNA #2, it was chosen for subsequent studies (Fig. 1B). As shown in Fig. 1, Bmi-1 was significantly silenced both at the mRNA and protein levels (Fig. 1B-D).

Significant morphological differences were observed in the A375-Bmi-1-shRNA cells in that there were more cohesive cells with epithelioid changes compared to the spindle-shaped normal A375 cells (Fig. 1E). The A375-Bmi-1-shRNA cells gathered more closely to each other, while the normal A375 cells appeared as spindle-like with a fibroblastic morphology (Fig. 1E). Meanwhile, Matrigel invasion chamber assays revealed significantly reduced invasion of the A375-Bmi-1-shRNA cells when compared to the control cells (Fig. 2D and E).

EMT marker expression following the silencing of Bmi-1 in A375 cells was detected and showed increased expression of epithelial markers (E-cadherin and α-catenin) with concomitantly decreased expression of mesenchymal markers (vimentin and N-cadherin) compared to the A375 control cells (Fig. 2A-C and F). Thus, our results suggested that Bmi-1 knockdown was crucial to reverse EMT-like changes and suppress invasiveness of cultured A375 cells in vitro.

To further evaluate the effect of Bmi-1 knockdown on tumor growth and EMT properties in vivo, six-week-old female BALB/c-nude mice were injected with the A375-vector and the A375-Bmi-1-shRNA cells in their flanks (six mice for each group). Both the A375-vector cells and the A375-Bmi-1-shRNA cells formed palpable tumors after 10 days, however, there was no marked difference between the two groups until the third week (Fig. 3B). Tumors in the control group grew quickly in the fourth and fifth week, while tumors in the A375-Bmi-1-shRNA group grew significantly slower with no visible substantial changes (Fig. 3B). The mice were observed for five weeks and the xenograft tumors were harvested. The tumor nodules formed by the A375-Bmi-1-shRNA cells were significantly smaller than those formed by the A375 control cells at the end of the experiment (day 35) (Fig. 3A and B). As the data showed (Fig. 3B and C), the average volume (A375-vector = 598.95±361.11 mm³, A375-Bmi-1-shRNA = 192.17±164.26 mm³, n=6, P=0.031) and the average of the weight (A375-vector = 624.2±246.8 mg, A375-Bmi-1-shRNA = 89.67±65.28 mg, n=6, P=0.000) had significant differences between the two groups.

To confirm that the silencing of Bmi-1 suppresses melanoma proliferative ability by reversing EMT tumorigenicity in mice, we evaluated the expression of EMT markers in tumor xenograft tissues. Compared with tumors formed by the A375-vector cells, the expression levels of E-cadherin and α-catenin were upregulated, while vimentin and N-cadherin were downregulated in the tumors formed by the A375-Bmi-1-shRNA cells (Fig. 3D). The findings were consistent with the gene expression of EMT markers in the Bmi-1-silenced A375 cells.

The tumor-suppressor PTEN, a downstream target of Bmi-1, was increased in the A375-Bmi-1-shRNA cells, while its targeted downstream molecules p-Akt, p-NF-κB and matrix metalloproteases-2 (MMP-2) were reduced when Bmi-1 was knocked down (Fig. 4). On the one hand, activation of Akt activated the NF-κB pathway, which promoted EMT in melanoma. On the other hand, the hyperactivated NF-κB pathway contributed to EMT properties by enhancing MMP-2 syntheses in melanoma. These results demonstrate that the silencing of Bmi-1 suppressed melanoma invasion via the regulation of PTEN/Akt/NF-κB/MMP-2 in A375 cells.