The Ubiquitin Ligase RNF8 On Immune Microenvironment Of Melanoma By Regulating Galectin-3

Objective:RNF8 is an E3 ubiquitin ligase,which has been shown to play a crucial role in DNA damage repair in previous studies.However,the role of RNF8 in tumor microenvironment remains unclear.The tumor microenvironment（TME）is crucial for initiation and development of tumor,which is composed of cancer cells,immune cells,stromal cells,the extracellular matrix and other components.Through multi-omics analysis,we found the downstream key proteins,galectin-3 can bind to many glycosylated molecules and plays an important role in the development and progression of tumor.However,there are still few reports on the mechanism of galectin-3 post-translational modification,especially the ubiquitination modification.Therefore,this study focused on the regulatory mechanism of RNF8 on ubiquitination modification of galectin-3.This study focused on the effect of the deletion of RNF8 in recipient mice on the tumor microenvironment of melanoma implant tumor,and explored the specific mechanism of the regulation of the tumor microenvironment of melanoma by RNF8.Methods:The gene deficient embryonic stem（ES）cell clone RRR260 was used to produce the RNF8^-/-mice.Overexpression and knockdown of RNF8 in cells with the help of lentivirus and plasmid.Transfection efficiency was verified by immunofluorescence and western-blotting.The interaction between galectin-3 and RNF8 was confirmed by co-immunoprecipitation,and its relationship was confirmed by immunofluorescence and western blot in the overexpressed and knockdown RNF8cell lines.The ubiquitination modification site of galectin-3 was predicted by protein modification omics data and mass spectrometry,and the site was verified by co-immunoprecipitation.By staining with histological examination（HE）and the specific markers of melanoma,the differences of tumor microenvironment in WT and KO group were compared.And the infiltration of immune factors and immune cells in the tumor microenvironment were detected by mass spectrometry（Cy TOF）.T-SNE and SPADE were used to analyze the subpopulations and numbers of immune cells.These results were verified by western blot and immunohistochemistry.Results:1.By comparing the rate of tumor growth,ratio of tumor weight and HE staining in WT and KO mice,we found that the rate of tumor growth was faster,ratio of tumor weight was larger,infiltration of immune cells and ratio of tumor stromal were less in KO mice.2.The results of mass spectrometry,immunohistochemistry and Western blot showed that the deletion of RNF8 in the recipient mice led to the decrease of lymphocyte infiltration in the tumor and the decrease of cytokine level such as IFN-γ,CXCL-9 and CXCL-10.In addition,there were significant differences in the distribution of lymphocytes and cytokines in the central and peripheral parts of melanoma.3.The interaction between RNF8 and galectin-3 was verified by molecular docking,co-IP and immunofluorescence methods,and the expression of RNF8 was negatively correlated with galectin-3.4.Immunoprecipitation results showed that RNF8 was ubiquitinated to Galectin-3 by K48-linked polyubiquitin chain.5.After the intervention of galectin-3 inhibitor Lac NAc,lymphocyte infiltration and cytokine levels in the tumor microenvironment of melanoma were increased,and the differences in the distribution of lymphocytes and cytokines in the central and peripheral parts of the tumor were improved.Conclusion:The deletion of RNF8 in recipient mice can accelerate the growth and progression of subcutaneous melanoma,and reduce the level of cytokines and the infiltration of immune cells in the melanoma microenvironment.In this study,it was proved that the expression of RNF8 was negatively correlated with the expression of galectin-3,after the binding of RNF8 to galectin-3,and the expression of galectin-3was modified by K48-linked polyubiquitination.Inhibition of galectin-3 restored the levels of cytokines and the infiltration of immune cells in the melanoma microenvironment.This study provides a deeper understanding of the ubiquitination pathway of galectin-3 and may provide a potential therapeutic option for the treatment of melanoma.