Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Dec 23:12:e18730.
doi: 10.7717/peerj.18730. eCollection 2024.

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

Ying Zhang  1 , Fengrui Cheng  1 , Xingrui Cai  2 , Jingping Wu  1
Affiliations

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

Ying Zhang et al. PeerJ. .

Abstract

Background: Epstein-Barr virus induced gene 3 (EBI3), a member of the IL-12 family, is known to be involved in malignant progression in a variety of cancers, but its role in melanoma is unclear. The aim of this study was to explore the effects of EBI3 on the malignant phenotype melanoma to reveal its potential as a therapeutic target.

Methods: In this study, we used bioinformatics to analyze the expression of EBI3 in pan-cancer and verified its expression level in melanoma cells by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Subsequently, the effects of EBI3 knockdown on cell proliferation, migration and invasion were detected using the Cell Counting Kit-8 (CCK-8) and Transwell assays. Changes in immune-related cytokines were detected by ELISA, and macrophage polarization was observed using immunofluorescence. Finally, the phosphorylation levels of signaling pathways such as Smad3, STAT6 and cGAS-STING were analyzed by Western blot.

Results: EBI3 was evidently highly-expressed in melanoma, and silencing of EBI3 could visibly suppress the survival and migration/invasion of melanoma cells, concurrent with the increased levels of BAX and CDH1 and the decreased expressions of BCL2 and CDH2. Meanwhile, EBI3 knockdown diminished the phosphorylation levels of both Smad3 and STAT6 and the levels of immune response-relevant cytokines in melanoma cells, while aggravating the macrophage M1 polarization and the expression of cGAS, p-STING and p-IRE1 α in THP-1 monocyte-derived macrophages co-cultured with EBI3-silenced melanoma cells.

Conclusion: This study filled the blank on the involvement of EBI3 in melanoma, hinting the possibility of controlling EBI3 as a therapeutic strategy in the management of melanoma.

Keywords: Epstein-Barr Virus Induced Gene 3; Immune response; Macrophage; Malignant behaviors; Melanoma.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Pan-cancer EBI3 expression analysis.
(A) Pan-cancer EBI3 expression analysis based on the data from TCGA. (B) Expression analysis on EBI3 level in skin cutaneous melanoma. SKCM, Skin cutaneous melanoma.
Figure 2
Figure 2. Effects of EBI3 knockdown on the viability of melanoma cells.
(A) The mRNA expression analysis on EBI3 in melanoma cell lines (M14, A2058, HMY1, MV3, and A875). (B) Validation on the transfection of EBI3-specific small interfering RNAs in melanoma cells via RT-qPCR. (C) Effects of EBI3-specific small interfering RNAs on the viability of melanoma cells via CCK-8 assay. ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. SKCM, Skin cutaneous melanoma.
Figure 3
Figure 3. Effects of EBI3 silencing on the migration, invasion and apoptosis modulators in melanoma cells.
(A and B) Transwell migration/invasion assay evaluating the migration and invasion of EBI3-silenced melanoma cells MV3 at 48 h. (C) Relative mRNA levels of metastasis-related cadherins (CDH1 and CDH2) in EBI3-silenced melanoma cells MV3. (D) Relative mRNA levels of apoptosis-related genes BAX and BCL2 in EBI3-silenced melanoma cells MV3. *p < 0.05; **p < 0.01.
Figure 4
Figure 4. Effects of EBI3 knockdown on the phosphorylation of SMAD3 and STAT6 as well as immune response in melanoma cells.
(A–C) Quantification on the phosphorylation of SMAD3 and STAT6 in EBI3-silenced melanoma cells MV3 via western blotting. (D) Measurement on the immune response-related cytokines IL-35, IL-4 and IL-13 in EBI3-silenced melanoma cells MV3 via ELISA. *p < 0.05; **p < 0.01.
Figure 5
Figure 5. Effects of EBI3 knockdown on the macrophage polarization.
(A) Scheme for immunofluorescence assay. (B and C) Mean fluorescence intensity of CD86 in these co-cultured cells. (D and E) Mean fluorescence intensity of CD206 in these co-cultured cells. *p < 0.05.
Figure 6
Figure 6. Effects of EBI3 knockdown on the macrophage polarization-related markers.
(A and B) The protein expression or the phosphorylation level of macrophage polarization-related markers (STING, c-GAS and IRE1α in THP-1 monocytes-derived macrophages following the co-culture with EBI3-silenced melanoma cells. *p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Ahmed B, Qadir MI, Ghafoor S. Malignant melanoma: skin cancer-diagnosis, prevention, and treatment. Critical Reviews in Eukaryotic Gene Expression. 2020;30(4):291–297. doi: 10.1615/CritRevEukaryotGeneExpr.2020028454. - DOI - PubMed
    1. Amuthalakshmi S, Sindhuja S, Nalini CN. A review on PCR and POC-PCR—a boon in the diagnosis of COVID-19. Current Pharmaceutical Analysis. 2022;18(8):745–764. doi: 10.2174/1573412918666220509032754. - DOI
    1. Awasthi A, Carrier Y, Peron JPS, Bettelli E, Kamanaka M, Flavell RA, Kuchroo VK, Oukka M, Weiner HL. A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells. Nature Immunology. 2007;8(12):1380–1389. doi: 10.1038/ni1541. - DOI - PubMed
    1. Azumi J, Takeda T, Shimada Y, Zhuang T, Tokuji Y, Sakamoto N, Aso H, Nakamura T. Organogermanium THGP induces differentiation into M1 macrophages and suppresses the proliferation of melanoma cells via phagocytosis. International Journal of Molecular Sciences. 2023;24(3):1885. doi: 10.3390/ijms24031885. - DOI - PMC - PubMed
    1. Bardi GT, Smith MA, Hood JL. Melanoma exosomes promote mixed M1 and M2 macrophage polarization. Cytokine. 2018;105(11):63–72. doi: 10.1016/j.cyto.2018.02.002. - DOI - PMC - PubMed

MeSH terms