| Part I:Expression and clinical significance of ubiquitin ligases Cbl-b in cutaneous melanoma tissue and three cell linesObjective:To investigate the expression of Cbl-b in cutaneous malignant melanoma(CMM)tissue and three cell lines(A375,M14 and MV3)and to assess the relationship of Cbl-b expression with the clinicopathological features of CMM.Methods:Immunohistochemistry was carried out to quantify the expression of Cbl-b in paraffin-embedded tissue samples from 69 cases of CMM and 30 cases of nevi.Quantitative real-time fluorescent PCR?Immunofluorescence and Western blot were respectively performed to detect the mRNA expression and protein expression of Cbl-b in melanocytes and three cell lines.Results:Immunohistochemistry revealed that the expression rate of Cbl-b was 75.36%(52/69)in melanoma tissue,compared to 13.33%(4/30)in nevus tissue(X2=32.745,p<0.01).The expression of Cbl-b increased sequentially from in situ melanoma to invasive and metastatic melanoma(rs=0.569,p<0.01).Additionally,the expression of Cbl-b was positively correlated with Clark level(rs=0.654,p<0.01)and Breslow depth(rs=0.727,p<0.01),and Cbl-b expression of ulcerated melanomas was significantly higher than that of non-ulcerated(p<0.01).There was significant difference in the expression of Cbl-b mRNA among three melanoma cell lines and melanocytes(F=176.537,p<0.01).Immunofluorescence and Western blot analysis showed the expression of Cbl-b protein could be all detected among the three melanoma cell lines and melanocytes,with the strongest level in A375 and the lowest level in melanocytes.Conclusion:The overexpression of Cbl-b protein may be related to the invasion and progression of melanoma.Part?:Construction of a lentiviral vector of Cbl-b shRNA and its effect on the biological behavior of melanoma A375 cells in vitroObjective:To construct a recombinant lentiviral vector of Cbl-b short hairpin RNA(shRNA)and explore its effect on the biologic behavior of melanoma A375 cells.Methods:Targeting human Cbl-b mRNA coding sequence,we designed three specific shRNAs,then constructed the recombinant lentiviral vector to transfect melanoma A3 75 cells.Quantitative real-time fluorescent PCR and Western blot were used to screen the best silencing shRNA.Cell proliferation was evaluated using cell counting kit-8(CCK-8).Cell apoptosis and cell cycle were analyzed by flow cytometry.Invasive potential was assessed by Transwell invasion assay.Results:Three recombinant lentiviral vector containing Cbl-b shRNA were constructed successfully.The CBLB-shRNA-3 showed the highest silencing efficiency.Compared with negative control(NC)and blank groups,the proliferation of A375 cells in the CBLB-shRNA-3 group was significantly decreased at 72h and 96 after transfection(p<0.01).The cell apoptotic rate of NC,blank and CBLB-shRNA-3 groups were(6.08±1.35)%,(6.34±1.07)%,(22.73±6.58)%,respectively,showing that the cell apoptotic rate of CBLB-shRNA-3 group was significantly higher than that of the NC and blank groups(p<0.01).Compared with the NC and blank groups,the percentage of cells in G1 phase was significantly increased in CBLB-shRNA-3 group(p<0.01),whereas the percentage in S phase was significantly decreased(p<0.01).The cell cycle was arrested at G1 phase.Transwell test showed that the number of penetrating cells in the NC,blank and CBLB-shRNA-3 groups were(76.60±1.82),(73.20±3.83),(19.60±1.14),with a significant difference(p<0.01).Conclusions:We have successfully constructed a recombinant lentiviral vector which can efficiently silence Cbl-b gene.Cbl-b gene silencing could inhibit proliferation,cell cycle progression,migration and promote apoptosis of A375 cells.Part ?:Proteomics analysis of Cbl-b-associated proteins in the A375 melanoma cellsObjective:To explore the relationship between Cbl-b and related proteins in the A375 melanoma cells.Methods:We performed comparative proteomics analysis by label-free to explore differentially expressed proteins in A375 cells with and without Cbl-b shRNA.Then,the properties of differentially expressed proteins were analyzed by the bioinformatics method.The expression of EphA2?GSK3? and p-AKT were detected by the Western blot after Cbl-b was knocked-down in A375 cells.Results:A total of 3449 proteins were identified and quantified,of which 74 proteins were significantly changed(with |Ratio|>2.0 or<0.5,p<0.05)between the two groups.Among them,52 proteins were overexpressed in the shRNA group compared with the NC group,and 22 proteins were downregulated.Cluster analysis found the shRNA and the NC group can be correctly classified by differentially expressed proteins.GO enrichment analysis revealed the main biological process included cell adhesion mediated by integrin?single-organism metabolic process?regulation of cell adhesion mediated by integrin?regulation of protein targeting to mitochondrion?nucleotide metabolic process and the main molecular function included DNA binding?2 iron,2 sulfur cluster binding?signaling receptor activity?cadherin binding.While KEGG enrichment analysis demonstrated the tumor-related signal pathways included folate biosynthesis?axon guidance?ECM-receptor interaction?adherens junction?Wnt signaling pathway?Hippo signaling pathway?antifolate resistance?oxidative phosphorylation?PI3K-Akt signaling pathway.EphA2 and GSK3? were confirmed by Western blot which were consistent with the proteomics results.The expression of p-AKT was downregulated after Cbl-b was silenced in A375 cells.Conclusion:Cbl-b may be involved in the pathogenesis of melanoma through a variety of signal pathways.The EphA2/PI3K/AKT signal pathway activated by Cbl-b may be one of the important pathogensis of melanoma. |
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