| Background: Melanoma is highly metastatic and has the highest mortality among skin cancers.The incidence rate has steadily increased over the past few decades,which poses a major challenge to the global health care system.Early melanoma patients can achieve better prognosis through surgical resection and radiotherapy and chemotherapy.However,the mortality rate of patients with advanced melanoma is still high.Although some progress has been made in the research of targeted drugs for melanoma,drug resistance is very common.Therefore,it is crucial to further explore potential therapeutic targets and develop new therapeutic strategies.Ebp1 was identified as ERBB3 receptor binding protein 1,encoded by PA2G4 gene,and widely expressed and localized in nucleus and cytoplasm.And Ebp1 is a cell cycle associated protein.Studies have shown that Ebp1 plays a variety of important roles in cell proliferation and differentiation.It is reported that the expression of Ebp1 is increased in glioblastoma,hepatocellular carcinoma and acute leukemia,and the high expression of Ebp1 is closely related to the poor prognosis.However,the role of Ebp1 in melanoma has not been reported.Therefore,exploring and studying the biological function of Ebp1 in melanoma is of great significance to further develop targeted drugs for the treatment of melanoma.Objective: This study mainly analyzes the role of Ebp1 in the occurrence and development of melanoma and its possible mechanism,to clarify the impact of Ebp1 on the clinical characteristics and prognosis of patients with melanoma,and to provide a new basis for exploring the potential therapeutic targets of melanoma.Methods: Part Ⅰ:(1)Observe the expression of Ebp1 in normal skin tissue and melanoma tissue through TCGA,GTEx and GEO databases.(2)The expression of Ebp1 in melanoma and normal skin was observed by immunohistochemical staining.(3)According to the clinical data of melanoma patients downloaded from TCGA database,the relationship between Ebp1 and the clinical characteristics and prognosis of melanoma patients was analyzed.Part II:(1)The expression of Ebp1 was specifically knocked down in A375 and B16 cells by lentivirus transfection.The effect of Ebp1 on the proliferation of melanoma cells was observed by CCK-8 assay,plate clone colony formation assay,soft agar colony formation assay and cell cycle assay.(2)The effect of Ebp1 on the migration ability of melanoma cells was analyzed by scratch test and Transwell test.(3)A375sh NC and sh Ebp1 cell lines were used to construct the tumor forming model in nude mice,and the expression of Ebp1 in the tumor was detected by immunohistochemical staining to analyze the effect of Ebp1 on the tumor forming ability in nude mice.(4)B16sh NC and sh Ebp1 cell lines were used to construct the lung metastasis model in nude mice.The lung metastasis in vivo was observed by H&E staining to study the effect of Ebp1 on the metastasis ability of melanoma cells in vivo.Part III:(1)The expression correlation between Ebp1 and EMT related proteins was predicted by GSEA enrichment analysis and GEPIA online website.(2)The expression levels of EMT related proteins in A375 sh NC,A375 sh Ebp1,B16 sh NC and B16 sh Ebp1 cells were detected by Western blot,and further verified by cellular immunofluorescence assay.(3)Predicting the correlation of Ebp1 and Wnt/β-catenin signaling pathway related proteins through GEPIA website.(4)Western blot was used to detect Wnt/β-catenin signaling pathway related proteins in A375 sh NC,A375 sh Ebp1,B16 sh NC and B16 sh Ebp1 cells.(5)Wnt signal pathway activator was added to A375 sh Ebp1 and B16 sh Ebp1 cell culture medium to detect the effects of activator on EMT and Wnt/β-catenin signaling pathway on the expression of related proteins.Results: Part Ⅰ:(1)It was observed in TCGA and GTEx databases that the expression of Ebp1 in melanoma tissues was significantly higher than that in normal skin tissues.(P=7.8e-14)(2)By mining GEO database,the expression of Ebp1 in melanoma tissue was significantly higher than that in normal skin tissue in GSE15605 gene set(P=0.0144).(3)The high expression of Ebp1 in melanoma was observed by immunohistochemical staining.(4)Among melanoma patients with high Ebp1 expression,male patients,stage T4 patients and recurrent patients account for a high percentage.(5)The overall survival time of melanoma patients with high Ebp1 expression was significantly shorter than that of melanoma patients with low Ebp1 expression.Part II:(1)CCK-8 assay,plate colony formation assay and soft agar colony formation experiment observed that knocking down the expression of Ebp1 decreased the growth activity and proliferation of A375 and B16 cells.(2)Cell cycle detection showed that knockdown of Ebp1 expression caused cell arrest of A375 and B16 in G1 phase and inhibited the proliferation of melanoma cells.(3)The results of scratch assay and Transwell assay showed that knocking down the expression of Ebp1 decreased the migration ability of A375 and B16 cells.(4)It was observed that knockdown of Ebp1 inhibited the growth of subcutaneous tumors in nude mice.(5)The results of lung metastasis experiment in nude mice showed that the knockdown of Ebp1 inhibited the lung metastasis ability of melanoma cells in vivo.Part III:(1)The expression of Ebp1 was positively correlated with the expression of EMT related protein by GSEA enrichment analysis and GEPIA online website analysis.(2)Knockdown of Ebp1 in A375 and B16 cells decreased the protein expression of vimentin,N-cadherin,slug and snail,and up regulated the protein expression of E-cadherin.(3)The expression of vimentin decreased,and the expression of E-cadherin increased in A375 and B16 cells after Ebp1 knockdown.(4)The result of Ebp1 expression analyzed by GEPIA website was positively correlated with the expression of β-catenin、GSK3β and Cyclin D1.(5)The results of Western blot showed that β-catenin、p-GSK3β and Cyclin D1 protein expression decreased after knocking down Ebp1,and the change of protein expression caused by Ebp1 knocking down was reversed after adding Wnt/β-catenin activator.Conclusion: 1.Ebp1 is highly expressed in melanoma tissues,and its expression level is positively correlated with the poor prognosis of melanoma patients.It has the potential to be a prognostic marker for melanoma patients.2.Ebp1 promoted the proliferation and metastasis of melanoma cells.3.Ebp1 regulates EMT through the Wnt/β-catenin signaling pathway to promote the invasion and metastasis of melanoma cells. |
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