To Explore The Effects And Potential Mechanisms Of Disulfiram/Copper On Murine Melanoma B16F10 Cells Based On Bioinformatics

Objective: Screen DEGs and hub genes with melanoma by bioinformatics.To investigate the effects of DSF / Cu on the proliferation,apoptosis,cell cycle,migration and invasion of murine melanoma B16F10 cells.Explore the relationship between disulfiram/copper and key genes.Methods: GSE35389,GSE31879 and GSE47980 were downloaded from GEO database.The DEGs between melanoma and normal samples were screened.Conduct KEGG and GO enrichment analyses of DEGs.Construct PPI network.Screen hub genes and conduct survival analysis on hub genes.Gene TRIP13 with the highest node degree was selected from hub genes,and its expression in malignant melanoma was demonstrated through GEPIA and Oncomine.CCK-8 was used to detect the effect of DSF/Cu on the proliferation of B16F10 cells.B16 F10 cells were cultured in vitro.The experimental groups were divided into a blank control group(Control group,containing 0.05% dimethyl sulfoxide solution)and a DSF/Cu group(0.5?M,0.75?M,1?M DSF,0.5?M Cu).Observe the cell morphology of the B16F10 cells after 12 hours of treatment.Flow Cytometry was executed to evaluate the cell cycle distribution and apoptosis of the control group and the DSF/Cu groups after 24 hours of treatment.Transwell assay detected the migration and invasion of B16F10 cells before and after treatment.The atomic force microscope(AFM)was applied to detect the change of Young's modulus in the nucleus of B16F10 cells after 12 hours of DSF/Cu treatment.Bax,Bcl-2,Cleaved-caspase3,CDK1,Cyclin B1,MMP9 and TRIP13 expression levels were investigated by Western Blot.Results:1.457 DEGs were identified from GSE35389,GSE31879 and GSE47980,consisting 293 downregulated genes and 164 upregulated genes.GO and KEGG analysis showed that upregulated genes mainly enriched in cell cycle,mitotic metaphase plate congression and mitotic chromosome condensation,while downregulated genes mainly enriched in melanin biosynthetic process,melanocyte differentiation and glutathione metabolism.2.The PPI network of DEGs was consist of 387 nodes and 2145 edges.The number of upregulated genes were 151,while downregulated genes were 227.Top 10 hub genes were selected from module,TRIP13 has the highest nodes.Survival analysis of hub genes showed that patients with malignant melanoma with RAB32,DDX59 and VPS18 change had a lower overall survival rate,and patients with RAB32 and PDK2 change had a lower disease-free survival rate.GEPIA showed that TRIP13 displayed higher levels as compared with normal tissues.Also,Oncomine database showed that TRIP13 were overexpress in melanoma.3.DSF/Cu has obvious inhibitory effect on proliferation of B16F10 cells.After treated with DSF/Cu(0.75?M,1?M)for 12 hours,cell density decreased and cells become round and fall off.After treated with DSF/Cu(0.5?M,0.75?M,1?M)for 24 hours,G2/M phase arrest occurred in the B16F10 cells(P <0.05,P <0.05,P <0.01),the number of apoptosis in B16F10 cells increased compared with the control group(P <0.05,P <0.01,P <0.001).Transwell assay showed that the migration and invasion of B16F10 cells in DSF/Cu(0.5?M,0.75?M,1?M)groups were significantly inhibited(P <0.001).The results of atomic force microscopy revealed that the Young's modulus of B16F10 cells in DSF/Cu(0.5?M)group was significantly higher than that in the control group(P <0.01).That is,the cell elasticity and deformability of the B16F10 cells in the DSF/Cu group were significantly reduced.The expression of Bax and Cleavedcaspase3 were higher in DSF/Cu(0.5?M,0.75?M,1?M)groups,while Bcl-2,CDK1,Cyclin B1,MMP9 and TRIP13 were lower than those in the control group.The increase and decrease of protein level is directly proportional to the concentration of DSF/Cu.Conclusion:1.Bioinformatics technology has identified target genes related to malignant melanoma,and TRIP13 may be potential biomarker for the diagnosis and treatment of malignant melanoma.2.DSF/Cu has obvious inhibitory effect on proliferation,migration and invasion of B16F10 cells.3.DSF/Cu may induce apoptosis,G2/M phase arrest and decrease of MMP9 expression in B16F10 cells by down-regulating the expression of TRIP13,inhibiting its proliferation,migration and invasion.