| Objective:When tumor cells invade the body,the immune system recognizes tumor-associated antigens(TAAs)on the surface of the tumor cells and activates specific humoral and cellular immune responses to clear the invading tumor cells;at the same time,tumor cells can negatively regulate the body’s immune responses through various channels to avoid immune supervision.At the same time,tumor cells can negatively regulate the body’s immune response and escape immune supervision through a variety of mechanisms.Immune infiltration is important in tumorigenesis and development,as well as in the clinical prognosis of cancer patients.Malignant melanoma is a tumor that develops from melanocytes in the body’s skin,mucous membranes,and retina.Cutaneous melanoma manifests as pigmented lesions that change dramatically over months or years.Despite its low incidence,it is highly malignant and susceptible to metastasis,with a high lethality rate.Melanoma responds to immunotherapy more favorably than other solid tumors,making it a more desirable candidate for research in the field of tumor immunology.The IFI30(Interferon Gamma-Inducible Protein 30)gene encodes the Gamma-Interferon-Inducible Lysosomal Thiol Reductase(GILT),a 250 amino acid precursor with a molecular weight of approximately 35 k D.The mannose-6-phosphate receptor can activate the intracellular IFI30 precursor(mannose6-phosphate receptor).IFI30could reduce disulfide bonds and promote the unfolding of proteins that contain disulfide bonds.Its thiol reductase activity requires an acidic lysosome environment.Previously,Professor Xu Xiaoyan’s team discovered that IFI30 can undergo RNA editing mediated by ADAR1 and ADAR2,members of the Adenosine Deaminases Acting on RNA(ADARs)family,as evidenced by RNA editing from A to I resulting in the replacement of the threonine at position 223(Threonine,T)to alanine,A.(T223A).However,the role of IFI30 in the tumor microenvironment and whether RNA editing results in altered IFI30 function is unclear.Antigen-presenting cells process antigenic peptides and bind to MHC I and II,resulting in the activation of CD4~+and CD8~+T cells,a process required for the body to recognize,clear pathogens,and even kill tumor cells.Overexpression of IFI30 in melanoma cells enhances CD4~+T cell responses,but the mechanism of activation of CD8~+T cells in melanoma and ADAR-mediated editing of IFI30 and functional alterations are unclear.It is important to note that IFN-γinduced IFI30 expression in melanoma cells is not regulated by classical CIITA,but by STAT1.Furthermore,there is evidence that IFI30 inhibits PAX3 expression in advanced human metastatic melanoma and increases sensitivity to radiotherapy.Because of its unique cytotoxic effects,CD8~+T is widely used in tumor immunotherapy,and IFI30 processing of class I antigens containing disulfide bonds also contributes to effective activation of CD8~+T cells.However,it is unclear how IFI30 mediates MHC class I antigen presentation in tumor cells,and it is critical to investigate the mechanisms by which IFI30 is regulated to understand the process of antigen presentation,melanoma progression,and immunotherapy.Cathepsin are a group of proteases found in the intracellular(especially the lysosomal fraction)of various animal tissues and are the main members of the cysteine protease family,which are closely associated with several major diseases such as human tumors,osteoporosis,and arthritis,and are a group of target proteases that have received much attention in recent years.Cathepsin are all made by the hydrolysis of inactive procathepsin,which consists of a signal-peptide,a propeptide and a catalytic domain containing the active center of the mature protease.The signal-peptide is between 10 and 20 amino acid residues in length and is responsible for transporting the ribosomally expressed procathepsin to the endoplasmic reticulum,where it is hydrolysed to form a procathepsin containing only the precursor peptide and the catalytic domain.The IFI30 and Cathepsin are regulated by each other.As mentioned earlier,cathepsin is involved in the maturation of IFI30 and different histases have different effects on the cleavage of IFI30 precursor peptides.Similarly,in melanoma cells IFI30 co-localized with Cathepsin B and D,but the underlying mechanism of regulation between IFI30 and Cathepsin B and D remains unclear.Cathepsin mediate the degradation of invariant chain li,allowing appropriate exposure of the antigen-binding furrow of the MHC molecule and thus determining the efficiency of antigen presentation.Exploring the regulation of cathepsin by IFI30 and the ability of IFI30itself to process peptides has helped us to understand the role of IFI30 in the MHC signalling pathway.In addition,Tumor-Associated Macrophages(TAM)are an important component of the tumor immune microenvironment,infiltrating most solid tumors in large numbers and promoting tumor progression by stimulating tumor cell proliferation,angiogenesis,metastasis and protecting tumor cells from immune system a.Therapeutic approaches to TAM can be broadly classified into two categories:TAM removal and TAM reprogramming,that is,inhibiting infiltrated macrophages or change its polarization.In general,tumor cells can secrete chemokines to recruit circulating macrophages,and macrophages can also secrete cytokines to regulate the biological functions of tumor cells.Studying the interaction between tumor cells and macrophages can help us understand the mechanism of tumor immune tolerance and provide theoretical support for immunotherapy.In summary,we would like to explore the following key questions:1.To investigate whether IFI30 and RNA-edited IFI30 can alter the HLA-I-specific antigen presentation process.2.To elucidate whether IFI30 and RNA-edited IFI30 can affect the M1/M2polarization of macrophages and whether changes in IFI30 expression in tumor cells affect their recruitment ability to macrophages.3.To reveal whether RNA-edited IFI30on the stability of cathepsin B.Methods:1.A375 and A2058 were stimulated with human IFN-γand Cathepsin B-specific inhibitor CA-074,respectively,to detect IFI30,Cathepsin B and HLA I protein levels.2.Lentiviral vectors were used to construct stable transfected cell lines in human melanoma cells A375,A2058 and mouse melanoma cells B16F10 overexpressing control,wild-type IFI30 and edited IFI30 plasmids.3.Real Time-quantitative Polymerase Chain Reaction(RT-q PCR)was used to detect IFI30 m RNA expression in A375 and A2058 stable-transformed cell lines,and Western Blot(WB)was used to detect the levels of IFI30 and V5 tag proteins in A375 and A2058 stable-transformed cell lines,and the levels of 6-his tag protein in B16F10 stable-transformed cell line were detected.4.In vitro proliferation of each group of cells was measured by colony formation assay and CCK8 counting(Cell Counting Kit-8).5.The growth rate of each tumor cell line in vivo was compared using tumorigenesis assay in BALb/c-nu mice and subcutaneous tumor formation assay in C57BL/6 mice.6.Spleens of each group of tumor-bearing mice were surgically removed,photographed,and weighed.Magnetic-activated cell sorting(MACS)was used to isolate the spleen CD8~+T.7.Flow Cyto Metry(FCM)was used to analyze the number of splenic CD8~+T in each group of tumor-bearing mice.8.Immunofluorescence(IF)was used to analyze the spleen CD8~+T in each group of tumor-bearing mice.The tumor-infiltrating CD8~+T cells of each group of tumor-bearing mice were analyzed.9.The expression of MHC class I molecules on the membrane surface of each group of mouse melanoma cells was identified by flow cytometry after stimulation with murine IFN-γ.10.The expression levels of HLA class I molecules at different locations in melanoma cells were probed by cytoplasmaic/membrane protein isolation.11.The human melanoma cells A375were treated with the proteasome inhibitor MG132 and the protein synthesis inhibitor CHX to detect protein degradation of IFI30,Cathepsin B and HLA I.12.Stimulation of groups of A375 cells using Cathepsin B-specific inhibitor CA-074 to detect IFI30,Cathepsin B and HLA I protein levels.13.After stimulation of THP-1 apposition and differentiation into M0 macrophages via PMA,extraction of the A375 cell supernatant was co-cultured with M0 macrophages,and the M1/M2 polarization markers of macrophages were detected by q PCR,and the levels of IFI30 and Cathepsin B were detected by WB.14.The M1/M2 polarization markers of tumor-associated macrophages in each group of tumor-bearing mice were analyzed by immunofluorescence.15.The femurs and tibias of each group of tumor-bearing mice were surgically removed,and the bone marrow-derived macrophages of mice were isolated and cultured ex vivo.16.The primary mouse bone marrow-derived macrophages were isolated and co-cultured with splenic-derived CD8~+T cells,and the activation ratio of CD8~+T cells(selected from granulomycin B and CD8 double-positive cells)was detected by flow cytometry.17.Cytokines were used to induce macrophage M1 and M2 polarization in vitro,respectively.Tumor cell conditioned medium was collected and co-cultured with polarized macrophages,and the effect of different tumor cell conditioned medium on the migration ability of differently polarized macrophages was examined by Transwell assay.18.After stimulation of THP-1 apposition and differentiation into M0 using PMA,M0 was stimulated with the cathepsin B specific inhibitor CA-074 and IFI30 protein levels were measured.Results:1.Both wild-type IFI30 and RNA-edited IFI30 inhibited the growth of melanoma cells in vitro.In the subcutaneous tumorigenesis assay in BALb/c-nu mice,wild-type IFI30 inhibited melanoma cell growth in vivo compared with the control group,and RNA-edited IFI30 inhibited melanoma cell growth in vivo compared with the wild group;in the subcutaneous tumorigenesis assay in C57BL/6 mice,wild-type IFI30 promoted melanoma cell growth in vivo compared with the control group,and RNA-edited IFI30 inhibited melanoma cell growth in vivo.2.Compared to controls,wild-type IFI30 tumor-bearing mice have fewer tumor-infiltrating CD8~+T cells,whereas edited IFI30 tumor-bearing mice have more CD8~+T cells.3.Tumor-associated macrophages in wild-type IFI30 tumor-bearing mice were predominantly M2,whereas tumor-associated macrophages in edited IFI30 tumor-bearing mice were predominantly M1.4.In melanoma cells,wild-type IFI30 inhibited the expression of HLA class I molecules on the membrane surface while promoting the expression of cytoplasmic HLA class I molecules,whereas edited IFI30 had the opposite effect.Wild-type IFI30promoted the degradation of total HLA I protein in melanoma cells by non-proteasome pathway,while the edited IFI30 inhibited the degradation by inhibiting the HLA I proteasome pathway,thus maintaining the protein stability.More importantly,wild-type IFI30 promoted the degradation of HLA I protein on the surface of melanoma cell membrane,while the edited type IFI30 delayed the degradation of HLA I protein on the surface of melanoma cell membrane.6.IFI30,both wild-type and edited type,promotes the degradation of mature CTSB via the proteasome hydrolysis pathway,interfering with its protein stability.IFI30 regulates the expression of total HLA I protein through CTSB.8.wild-type IFI30 tumor supernatant inhibits macrophage M1polarization by inhibiting CTSB maturation in macrophages.9.wild-type IFI30 tumor supernatant inhibits M1 macrophage migration and promotes M2 macrophage migration mediated by CTSB,whereas edited-type IFI30 has the opposite effect.Conclusion:1.IFI30 inhibits tumor-infiltrating CD8~+T cells and reduces the M1polarization state of tumor-associated macrophages in melanoma cells.2.IFI30suppresses HLA I expression by blocking CTSB.3.IFI30 may affect CTSB and HLA I protein intracellular stability by promoting their degradation. |
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