| Part Ⅰ Explore the expression and function of αvβ3-integrin in melanomaObjective:Integrins are abundantly expressed in tumor cells and tumor stromal cells,and are involved in cancer development,invasion,and metastasis.This study aims to explore the expression and function of αvβ3-integrin in melanoma by bioinformatics methods.Methods:The clinical information and transcriptome sequencing data of 470 patients with melanoma were downloaded from The Cancer Genome Atlas(TCGA).Normal skin samples in the Genotype-Tissue Expression(GTEx)database were used as controls to compare the difference of αvβ3-integrin expression between melanoma and normal skin samples.Next,470 melanoma patients in TCGA were divided into the high αv-integrin expression group,low αv-integrin expression group and the high β3-integrin expression group,low β3-integrin expression group according to the median value of each gene.The differently expressed genes between the high and low expression groups of each gene were screened for pathway enrichment analysis.The correlation between αvβ3-integrin and immune checkpoint molecules was investigated by spearman correlation analysis.Finally,the relationship between αvβ3-integrin and the immune microenvironment of melanoma was studied by TIDE score and MCP-counter and XCELL algorithm.The response and prognosis of melanoma patients to immunotherapy were predicted at the same time.Results:αvβ3-integrin was highly expressed in melanoma compared with normal skin tissue samples in GTEx database.There were significant differences in PI3K/AKT and cell adhesion related pathways between the high αvβ3-integrin expression group and low αvβ3integrin expression group.β3-integrin was positively correlated with the expression of CD274,CTLA4、HAVCR2 and PDCD1LG2.αv-integrin was positively correlated with the expression of CD274,HAVCR2 and PDCD1LG2,and was negatively correlated with the expression of SIGLEC15.In addition,the TIDE score in the high β3-integrin expression group was higher than that in the low β3-integrin expression group.The infiltration of T cells was decreased in the the high β3-integrin expression group,but the infiltration of macrophages and endothelial cells was increased.There was no significant difference in TIDE score between the high αv-integrin expression group and low αv-integrin integrin expression group.The infiltration of T cells was decreased in the high αv-integrin expression group,but the infiltration of regulatory T cells,macrophages and endothelial cells was increased.No significant difference was observed in the infiltration of B cells among the above groups.Conclusion:The high expression of αvβ3-integrin in melanoma was found by bioinformatics methods.In addition,integrin αvβ3 was related to immune infiltration and the efficacy and prognosis of immunotherapy in melanoma.Therefore,integrin αvβ3 is expected to become a potential therapeutic target for melanoma.Part Ⅱ Explore the biological function of αvβ3-integrin antagonist cilengitide in melanomaObjective:Melanoma is one of the most aggressive skin cancers with a poor prognosis.αvβ3-integrin is an adhesion protein,which takes part in tumor growth,metastasis and angiogenesis.At present,little is known about the therapeutic effect and its potential mechanism of the αvβ3-integrin antagonist cilengitide on melanoma.This study aims to investigate the effect of cilengitide on the biological function of melanoma cells.Methods:Transcriptome sequencing was used to compare the down-regulated genes and functional enrichment pathways of B16 melanoma cells treated with cilengitide compared with normal B16 cells.CCK8 cell proliferation and toxicity assay and clone formation assay were used to explore the effect of cilengitide on the proliferation of melanoma cells.The proportion of apoptotic cells in melanoma cells treated with cilengitide was detected by flow cytometry.And the content of Cleaved Caspase-3 was detected by western blot.Then the effect of cilengitide on the invasion and migration of melanoma cells was detected by transwell and scratch test.Finally,epithelial mesenchymal transformation related proteins were detected by western blot.Results:PI3K/AKT and MAPK pathways were down-regulated in B16 melanoma cells treated with cilengitide compared with normal B16 cells.Cilengitide significantly inhibited the proliferation of B16 and A375 melanoma cells in vitro,and induced cell apoptosis by promoting the activation of Caspase-3.In addition,cilengitide inhibited TGF-β/Smad signaling pathway and inhibit epithelial mesenchymal transformation,weaken the invasion and migration ability of melanoma cells.Conclusion:αvβ3-integrin played an important role in regulating the biological function of melanoma cells.αvβ3-integrin antagonist cilengitide showed significant anticancer effect by inhibiting the proliferation and epithelial mesenchymal transformation process,and by inducing apoptosis of melanoma cells.Part Ⅲ Explore the effect of cilengitide combined with anti-PD1 antibody on murine melanoma modelObjective:Studies have shown that αvβ6-integrin and αvβ8-integrin inhibitors have synergistic effects when used in combination with immunotherapy.However,the role ofαvβ3-integrin in immune microenvironment of melanoma is unclear.This study aims to explore the effect of αvβ3-integrin antagonist cilengitide on the expression of PD-L1 and the efficacy of immune checkpoint inhibitors in melanoma.Methods:Immunofluorescence,flow cytometry and western blot were used to investigate the regulation and potential mechanism of cilengitide on the expression of PD-L1 in melanoma cells in vitro.In addition,the B16 murine melanoma model was constructed to verify the effect of cilengitide on PD-L1 expression in vivo.And we discussed the effect of the combination of cilengitide and anti-PD1 antibody on tumor growth and T cell function.Results:Cilengitide downregulated the expression of PD-L1 in melanoma in vitro and in vivo,which was correlated with the suppression of the binding of phosphorylated STAT3 to PD-L1 promoter.The combination of cilengitide and anti-PD1 antibody showed stronger antitumor effect than single-agent treatment in B16 murine melanoma model.The survival time of mice in the combined treatment group was prolonged.The proportion of CD8+T cell infiltration and the release of interferon-γ and granzyme B increased in tumor tissues.Conclusion:The combination of cilengitide and anti-PD1 antibody increased the infiltration of CD8+T cells.Combination therapy improved the sensitivity of immunotherapy,and resulted in significant tumor regression and longer survival in the B16 murine melanoma model. |
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