RNF43 Affects Melanoma Anti-tumor Immunity Via Immune Checkpoint PD-L1

Melanoma,usually referred to as malignant melanoma,is a highly malignant tumor derived from melanocytes,mostly in the skin,but also in the mucous membrane and internal organs,among which cutaneous melanoma is the most common skin cancer.Melanoma is a relatively rare malignant tumor in China,but its high mortality and high incidence in recent years deserve our attention.In recent years,the application of tumor immunotherapy such as immune checkpoint inhibitors has brought fresh ideas for the treatment of melanoma patients,but some patients still have problems such as low reactivity or drug resistance.Therefore,in the process of tumor immunotherapy,how to reduce the immune escape of tumor and promote the killing of immune cells to tumor cells is a difficult problem to resolve.As a member of the immune checkpoint proteins,programmed cell death 1 ligand1(PD-L1)can be expressed on many cancer cells and immune cells,and when it binds to programmed death 1(PD-1)on T cells,it can cause immune escape.This can be reflected in the inhibition of cytotoxic mediator secretion,proliferation and migration of T cells,and affects functions such as killing of tumor cells.Therefore,how to regulate the expression of PD-L1 in tumor cells or how to reduce the expression of PD-L1 in tumor cells is the question we want to explore.Through preliminary experimental screening,we found that ring finger protein 43(RNF43),an E3 ubiquitin ligase,can act through negative regulation of Wnt signaling pathway,and the expression level of RNF43 is often significantly increased or decreased in tumor tissues,with anti-apoptotic and pro-growth effects.Here we focused on the function of RNF43 negatively regulating Wnt signaling pathway,and some studies showed that Wnt/β-catenin signaling pathway in melanoma can lead to T cell rejection and resistance to immune checkpoint inhibitor treatment,causing immune escape.Therefore,we hypothesized that RNF43 may affect the melanoma tumor microenvironment through the regulation of Wnt signaling.Wnt/β-catenin signaling,also known asβ-catenin-dependent pathway or classical Wnt signaling pathway,mainly controls cell proliferation,which in whichβ-catenin is the main effector molecule of the Wnt signaling pathway.Different genetic alterations can inhibit the degradation ofβ-catenin by the proteasome,leading to excessive activation of classical Wnt signaling and enhanced nuclearβ-catenin accumulation.It has been shown that theβ-catenin/T cell factor(TCF)/lymphoid enhancer-binding factor(LEF)complex binds to the promoter region of the CD274 gene to induce PD-L1 expression in glioblastoma.We therefore hypothesized that in melanoma,β-catenin may play the same function,while RNF43 may affect PD-L1 expression through regulation ofβ-catenin.To clarify the regulatory role of RNF43 on PD-L1 in melanoma and the effect on anti-tumor immunity,we constructed RNF43 stable over-expressing and knockdown cell lines in the mouse melanoma cell line B16-OVA and carried out subsequent experiments based on this.Method:(1)Bioinformatics analysis was conducted by GEPIA and TIMER databases to explore the expression changes of RNF43 in melanoma patient tissues and the relationship between expression levels and patient survival,and the effect of RNF43 on the melanoma tumor microenvironment was also analyzed.(2)Melanoma cell lines with stable over-expressing and knockdown of RNF43were constructed by lentivirus infection of cells and puromycin screening,and the expression level of RNF43 protein was verified by Western blot.(3)The effect of RNF43 on melanoma cell proliferation in vitro and in vivo was detected by clone formation assay and subcutaneous tumor loading assay in mice,while mouse tumor infiltrating lymphocytes were isolated to explore the effect of RNF43 on melanoma immune cell infiltration.(4)The effect of RNF43 onβ-catenin and PD-L1 in different melanoma cell lines was detected by Western blot and real-time fluorescence quantitative PCR.(5)RNF43 andβ-catenin over-expressing plasmid and PD-L1 promoter luciferase reporter gene plasmid was constructed,and the regulatory relationship of RNF43 on PD-L1 was detected by dual luciferase reporter gene assay.Results:(1)GEPIA and TIMER analysis showed that the expression of RNF43 was decreased in melanoma patients,and patients with melanoma with high expression of RNF43 had better survival,and RNF43 may promote the infiltration of immune cells in the melanoma tumor microenvironment.(2)RNF43 stable over-expressing and knockdown melanoma cell lines Lv-RNF43-OE and LV-RNF43-KD was successfully constructed in this experiment by lentiviral packaging plasmid and puromycin screening and western blot.(3)The results of clone formation assay showed that RNF43 could inhibit the proliferation of melanoma cells in vitro;the results of subcutaneous tumor loading assay in mice showed that RNF43 could inhibit the proliferation of melanoma cells in vivo.(4)Flow cytometry results showed that RNF43could promote the release of CD8~+T cell perforin and interferonγ(IFN-γ)in the melanoma tumor microenvironment and promoted the killing effect on melanoma.(5)Western blot and real-time quantitative PCR showed that RNF43 could inhibitβ-catenin expression and PD-L1 m RNA expression in melanoma cells.(6)RNF43 andβ-catenin over-expressing plasmids and PD-L1 promoter luciferase reporter plasmids were successfully constructed in this experiment,(7)The Dual Luciferase reporter gene results suggested that RNF43 could reduce the transcription expression of PD-L1 in melanoma by inhibitingβ-catenin.Conclusion:RNF43 can reduce the transcription expression of PD-L1 in melanoma by inhibiting the expression ofβ-catenin and promote CD8~+T cell-mediated anti-tumor immune response.