Malignant behaviors and immune response in melanoma: Epstein-Barr virus induced gene 3 as a therapeutic target based on an in-vitro exploration

Cell culture

Human melanoma cell lines M14 (BNCC340599; BeNa Culture Collection, Beijing, China), A2058 (CL-0652; Procell, Wuhan, China), HMY1 (CL-0696; Procell, Wuhan, China), MV3 (BNCC339913; BeNa Culture Collection, Beijing, China) and A875 (BNCC359299; BeNa Culture Collection, Beijing, China) were applied for the quantification on EBI3 mRNA expression. Leibovitz’s L-15 medium (SH30525; Cytiva Lifesciences, Marlborough, MA, USA) or high-glucose Dulbecco’s modified Eagle’s medium (SH30022; Cytiva Lifesciences, Marlborough, MA, USA) with the supplementation of 10% bovine calf serum (SH30071; Cytiva Lifesciences, Marlborough, MA, USA) and 1% penicillin-streptomycin (SV30010; Cytiva Lifesciences, Marlborough, MA, USA). Human monocyte cell line THP-1 (BNCC358410; BeNa Culture Collection, Beijing, China) were additionally cultured in Roswell Park Memorial Institute-1640 medium (SH30255; Cytiva Lifesciences, Marlborough, MA, USA) plus 10% bovine calf serum, 1% penicillin-streptomycin and 0.05 mM β-mercaptoethanol (M301574; Aladdin, Shanghai, China). All cell lines were tested via STR method and free of mycoplasma contamination, and incubated in an incubator at 37 °C with 5% CO₂. For the induction into macrophages in vitro, THP-1 monocytes were induced with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, P408905; Aladdin, Shanghai, China) for 48 h, followed by the incubation in Roswell Park Memorial Institute-1640 medium (Phuangbubpha et al., 2023).

Cell transfection

The small interfering RNAs against EBI3 with specific target sequences (si EBI3-1 and si EBI3-2) as well as the control small interfering RNAs with random sequence were all synthesized and purchased from GenePharma (Shanghai, China, https://www.genepharma.com/), which were then transfected into melanoma cells with Lipofectamine 3000 transfection reagent (L3000-001; Thermo Fisher Scientific, Waltham, MA, USA) for 48 h at 37 °C as per the manuals. All cells were harvested and the transfection efficiency was tested via reverse-transcription quantitative PCR (RT-qPCR). The sequences for the transfection were listed in Table 1.

Cell proliferation test

Melanoma cells were counted and distributed in 96-well culture plates (2 × 10³/well) for the culture at the indicated time points. Then, the culture medium for melanoma cells was replaced with the one containing 10 μL CCK-8 solution (CK04; Dojindo Laboratories, Kumamoto, Japan). Melanoma cells were then continued for 4-h incubation in the incubator at 37 °C with 5% CO₂. The optical density (OD) value at the 450 nm was recorded in SPECTRO Star Nano microplate reader (BMG Labtech, Gary, NC, USA) and the viability of cells in each group was tested and plotted in a curve (Tian et al., 2023).

Cell migration/invasion test

Following the transfection for 48 h, 100 μL melanoma cells were harvested and inoculated into 24-well plates in the upper Transwell chamber (pore: 8 μm, 3422; Corning, Inc., Corning, NY, USA) coated with/out the Matrigel^® matrix (354230; Corning, Inc., Corning, NY, USA), which was added with 200 μL serum-free cell culture medium. In the meantime, 600 μL complete medium was added to the lower Transwell chamber. A total of 48 h after the culture, the migrated and invaded cells were fixed and dyed, followed by the quantification under an optical microscope (Nikon, Tokyo, Japan).

ELISA

IL-35 has a significant immunosuppressive effect and is able to modulate the function of T cells and macrophages (Ye et al., 2021). And two cytokines, IL-4 and IL-13, which are closely related to the polarization of M2-type macrophages, are able to promote the formation of an immunosuppressive microenvironment, which in turn supports tumor growth and metastasis (Shi et al., 2021; Bernstein et al., 2023). To be able to assess the role of EBI3 in regulating the immune microenvironment of melanoma, we chose IL-35, IL-4, and IL-13 as indicators for assessing cellular immune response. In this study, melanoma cells were cultured in the six-well culture plates at the density of 8 × 10⁵/well for 24 h. Hereafter, the cell culture supernatant was harvested for the calculation on the levels of relevant cytokines including IL-35 (NBP3-06774), IL-4 (NBP1-91171) and IL-13 (NBP1-91176) as per the manuals of the producer (Novus Biologicals, Centennial, CO, USA). The absorbance at 450 nm was read in the microplate reader.

Co-culture system preparation

Transwell chambers (pore: 0.4 μm, 3470; Corning, Inc., Corning, NY, USA) were applied and settled in a 24-well plate. Hereafter, 500 μL induced macrophages (3 × 10⁵/mL) were transferred to the upper chambers, while the lower chambers were filled with 1 mL melanoma cells (2 × 10⁵/mL). These plates were then put in the incubator for 8-h culture, as described elsewhere (Yan et al., 2022).

Cell immunofluorescence assay

Following the co-culture of melanoma and macrophages, the polarization of macrophages was tested in cell immunofluorescence assay. In detail, 4% fixative paraformaldehyde solution (P395744; Aladdin, Shanghai, China) and 0.5% Triton X-100 (T109026; Aladdin, Shanghai, China) were serially applied to fix and permeabilize the co-cultured cells, which were hereafter washed in PBS thrice (5 min for each time). Following the blocking in 5% goat serum (C0265; Beyotime Institute, Shanghai, China), cells were incubated with the following primary antibodies (Abs) against CD11b (Alexa Flour^® 488, 53-0112-82; Thermo Fisher Scientific, Waltham, MA, USA), CD86 (Alexa Flour^® 647, 105019; BioLegends, Inc., San Diego, CA, USA) and CD206 (Alexa Flour^® 647, MA5-44147; Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C. After the washing in cold PBS twice, co-cultured cells were incubated in goat-anti rabbit IgG secondary antibody (ab6702; Abcam, Cambridge, UK) at ambient temperature for 1 h in a dark room. Following the staining of the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, 62247; Thermo Fisher Scientific, Waltham, MA, USA), images of cells were captured in a fluorescence microscope (Nikon, Tokyo, Japan).

RNA extraction and reverse transcription-quantitative polymerase chain reaction

TRIzol reagent (15596026; Thermo Fisher Scientific, Waltham, MA, USA) was applied for the extraction of total cellular RNA. A total of 1 μg of the extracted RNA was reversely transcribed into complementary DNA (cDNA) using iScript™ cDNA synthesis kit (4106228; Bio-Rad, Hercules, CA, USA) as per the manuals. The qPCR was then carried out using iQ™ SYBR^® green supermix (1708880; Bio-Rad, Hercules, CA, USA) in CFX96 touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA) at the following cycling steps: 95 °C for 2 min and 40 repeated cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The primers applied are listed in Table 2. 2^−ΔΔCT method was applied for the calculation of the relative mRNA expressions with GAPDH as the housekeeping control (Livak & Schmittgen, 2001; Amuthalakshmi, Sindhuja & Nalini, 2022).

Protein extraction and western blotting analysis

RIPA lysis buffer (P0013B; Beyotime Institute, Shanghai, China) was adopted to lyse the total cellular protein from melanoma cells or induced macrophages containing the protease and phosphatase inhibitor cocktail (P1045; Beyotime Institute, Shanghai, China) and the equal amount of protein sample was loaded into and detached in sodium dodecyl sulfate-polyacrylamide gels for electrophoresis. Subsequently, the protein samples were transferred to the polyvinylidene fluoride membranes (FFP32; Beyotime Institute, Shanghai, China) (Zhang et al., 2023), which were then blocked in 5% western blotting buffer (P0252; Beyotime Institute, Shanghai, China) and incubated with the following primary Abs (Abcam, Cambridge, UK) against p-SMAD3 (ab74062, 1/1,000 dilution), SMAD3 (ab40854, 1/1,000 dilution), signal transducer and activator of transcription 6 (STAT6, ab32108, 1/5,000 dilution), p-STAT6 (ab263947, 1/1,000 dilution), cyclic GMP-AMP synthase (c-GAS, ab302617, 1/1,000 dilution), p-stimulator of interferon genes (STING, ab318181, 1/1,000 dilution), STING (ab239074, 1/1,000 dilution), p-inositol-requiring enzyme 1α (IRE1α, ab124945, 1/2,000 dilution), IRE1α (ab96481, 1/2,000 dilution) and housekeeping control GAPDH (ab8245, 1/10,000 dilution) at 4 °C overnight. Further, the membranes were reacted with horseradish peroxide-conjugated secondary Abs against rabbit IgG (ab205718; Abcam, Cambridge, UK) or mouse IgG (ab205719; Abcam, Cambridge, UK) at 1/5,000 dilution for 1-h incubation at ambient temperature.

Finally, for the visualization process, the membranes were sequentially rinsed in TBST buffer (ST671; Beyotime Institute, Shanghai, China) and exposed in BeyoECL Plus visualization reagent (P0018S; Beyotime Institute, Shanghai, China). The analysis was implemented in ChemiDoc™ touch imaging system (Bio-Rad, Hercules, CA, USA) and the densitometry on the grey value was carried out with the help of ImageJ 5.0 (Bio-Rad, Hercules, CA, USA).

Statistics

All statistical analyses were implemented applying GraphPad Prism 7 (GraphPad, Inc., La Jolla, CA, USA) and all data were compared with student’s t-test and presented as mean ± standard deviation. Changes at the significance level of p-value < 0.05 were assumed to be statistically significant.

Pan-cancer EBI3 expression analysis

With the purpose of exploring the involvement of EBI3 in melanoma, the pan-cancer EBI3 expression pattern was predicted and downloaded (Fig. 1A). Notably, skin cutaneous melanoma was screened out and the level of EBI3 was additionally predicted, and an elevated EBI3 expression was observed (Fig. 1B, p-value < 0.05). Therefore, in our subsequent assays, the corresponding small interfering RNAs with EBI3-specific targeting sequences were applied for the knockdown assays.

Effects of EBI3 knockdown on the malignant phenotypes of melanoma cells

Subsequently, the expression levels of EBI3 in melanoma cells were quantified in the beginning, and the highest expression of EBI3 was seen in MV3 cells (Fig. 2A, p-value < 0.01). Then the relevant small interfering RNAs with EBI3-specific targeting sequences were constructed and transfected into MV3 cells via liposome, and the successful knockdown of EBI3, as seen by the decreased EBI3 mRNA expression (Fig. 2B, p-value < 0.001). The relevant results from the Cell Counting Kit-8 (CCK-8) assay then suggested that silencing of EBI3 using these two specific small interfering RNAs could visibly diminish the viability of MV3 cells (Fig. 2C, p-value < 0.01).

Transwell migration/invasion assay was adopted to evaluate the migration and invasion of melanoma cells following the knockdown of EBI3. As shown in Figs. 3A and 3B, at 48 h, the reduced number of both migrated and invaded melanoma cells was clearly seen following the knockdown of EBI3 (Figs. 3A and 3B, p-value < 0.01). Also, to further explore the potential effects of EBI3 on epithelial-mesenchymal transition (EMT) signatures, we examined the mRNA expression levels of CDH1 and CDH2 upon EBI3 silencing. As shown in Fig. 3C, the silencing of EBI3 could contribute to the reduced expression of CDH2 yet the increased level of CDH1 (p-value < 0.05). Further, the expression levels of the apoptosis-related regulator BCL2 family proteins (BAX and BCL2) were quantified, and the silencing of EBI3 led to the suppressed BCL2 expression and the enhanced BAX expression in MV3 cells (Fig. 3D, p-value < 0.05). These findings further support the critical role of EBI3 in promoting the malignant behavior of melanoma; not only does EBI3 play a facilitating role in tumor cell migration, invasion, and survival, but its knockdown not only inhibits the EMT process but also induces apoptosis.

Effects of EBI3 knockdown on the phosphorylation of SMAD3 and STAT6 in melanoma cells

The activation of two pathways, TGF-β and IL-4/IL-13, play important roles in malignant behaviors such as tumor cell growth, migration, and immune response; therefore, by investigating the phosphorylation levels of Smad3 and STAT6 (Zeng et al., 2024; Hu et al., 2024), it is possible to further understand whether EBI3 can have an effect on the presence of melanoma through these pathways. In this study, we focused on Smad3 and STAT6 in melanoma cells, and the phosphorylation of these two proteins was determined in western blotting assay accordingly. The relevant quantification results in this study demonstrated that the phosphorylation of both Smad3 and STAT6 was obviously diminished by EBI3-specific small interfering RNA (Figs. 4A–4C, p-value < 0.05).

Effects of EBI3 knockdown on the immune response in melanoma cells

ELISA was used to investigate the potential effects of EBI3 knockdown on the immune response of melanoma cells via calculating the concentrations of specific cytokines. The relevant results have demonstrated that the concentrations of these specific cytokines including IL-35, IL-4 and IL-13 were all higher in melanoma cells with the presence of EBI3-specific small interfering RNA (Fig. 4D, p-value < 0.05).

Effects of EBI3 knockdown on the macrophage polarization

Additionally, the effects of EBI3 knockdown on the macrophage polarization were further explored, and the scheme for immunofluorescence assay was listed in Fig. 5A. Then the corresponding results from immunofluorescence assay has suggested that the knockdown of EBI3 in melanoma cells contributed to the reduced CD206 fluorescence intensity yet the increased CD86 fluorescence intensity in these induced macrophages from THP-1 monocytes (Figs. 5B–5E, p-value < 0.05).

The levels of relevant macrophage polarization indicators have been further quantified via western blotting. It was seen in THP-1 monocytes-induced macrophages that the silencing of EBI3 in melanoma cells could visibly upregulate the expression of c-GAS and promote the phosphorylation of both STING and IRE1α (Figs. 6A and 6B, p-value < 0.05). This suggests that knockdown of EBI3 activates the cGAS-STING and IRE1α signaling pathways in THP-1-derived macrophages, which may be associated with enhanced immune response and pro-inflammatory status and contribute to the polarization of M1-type macrophages.