WD Repeat And SOCS Box Containing Protein 2 In The Proliferation And Migration Of Melanoma Cells

Part ?:The relationship between WSB2 expression and clinicopathological features in melanomaObjective:In the previous study,WSB2 screened by mRNA array is an oncogene specifically expressed in melanoma.Here,bioinformatics analysis,immunoblotting and immunohistochemistry were used to explore the expression of WSB2 in melanoma tissues.It is found that WSB2 expression is related to clinicopathological features and prognosis of the patient.It provides a scientific basis for WSB2 to become a molecular target in the diagnosis and treatment of melanoma.Methods:First,the bioinformatics analysis was used to evaluate the expression of WSB2 in the NCBI database and the TCGA database.The relationship between the survival of tumor patients.A retrospective study was conducted on 49 cases of malignant melanoma and 49 cases of benign nevi tissues in the Third Affiliated Hospital of Kunming Medical University(Yunnan Cancer Hospital)from January 2010 to December 2015.Immunohistochemistry and western blot were used to evaluate the positive expression of WSB2 in human melanoma and pigmented nevi,and to analyze the relationship between WSB2 positive expression and clinicopathological features in melanoma patients.Patients with malignant melanoma were followed up.The relationship between WSB2 positive expression and overall survival were assessed One-way anova analysis,t-test,chi-square test,and Kaplan-Meier survival analysis were used in the studyResults:? Bioinformatics analysis showed that 70 cases of tissue specimens were included in the study using NCBI database,including 45 cases of melanoma,18 cases of nevi,7 cases of normal skin.WSB2 in melanoma expressed higher than nevi and normal skin(p<0.05);? Bioinformatics analysis showed that patients with melanoma with high expression of WSB2 were more likely to have shorter overall survival than those with low expression(p<0.05).? Collecting melanoma and nevi tissue from inpatients of the Third Affiliated Hospital of Kunming Medical University(Yunnan Cancer Hospital),using westernblotting to show high expression of WSB2 protein in melanoma tissues,with higher gray value(p<0.05);? Retrospective analysis showed that from January 2010 to December 2015,49 patients with malignant melanoma and 49 patients with nevi in the Third Affiliated Hospital of Kunming Medical University(Yunnan Cancer Hospital)were enrolled;? Immunohistochemistry results showed that the positive expression rate of WSB2 in melanoma was higher than that of nevi(69.39%vs.38.78%,p<0.05);? The relationship between the expression of WSB2 and the clinicopathological features of patients:the positive rate of WSB2 was significantly increased in patients aged 60 years and older(86.36%vs.51.85%,p=0.015);the positive rate of WSB2 in MM was significantly higher than that in CM(91.67%).Vs.57.14%,p=0.037);the positive rate of WSB2 in lymph node-positive patients was significantly higher than that in node-negative patients(100%vs.61.67%,p<0.05).? Kaplan-Meier survival analysis showed that patients with high expression of WSB2 in skin melanoma had shorter overall survival than those with low expression(p<0.05).Conclusion:WSB2 may have important clinical significance of being an important molecular marker for predicting the development and progression of melanoma,malignancy,prognosis and specific pathological types.Part ?:The biological function of WSB2 in vitro and in vivoObjective:In the first part of the study,it was found that WSB2 was highly expressed in melanoma,and led to a poor prognosis.The effects of WSB2 on cell proliferation,migration,apoptosis and cell cycle in melanoma cell lines A375 and G361 were investigated to confirm the clinical and pathological features The mouse xenografts experiment was further used to explore the effect of WSB2 on melanoma in vivo.It is intended to obtain reliable evidence that WSB2 promotes malignant processes such as tumorigenesis and development,and provides a scientific basis for WSB2 to become a new target for melanoma treatment.Methods:First,the protein expression of WSB2 was detected in a human melanoma cell line by westernblotting.Then,A375 and G361 cells were infected with WSB2-shRNA lentiviral interference vector to knock down the WSB2 gene,which reduced the expression of WSB2.After inhibiting the expression of WSB2,the cell viability was measured by MTS assay,and the effect of WSB2 on the proliferation of melanoma cells was evaluated.After inhibiting the expression of WSB2,the colony formation assay was performed.Cell cycle and apoptosis were detected by flow cytometry after the expression of WSB2 inhibited.Finally,mouse transplant tumor experiments were used to evaluated the effects of WSB2 on melanoma growth and metastasis in vivo One-way anova analysis,t-test.and chi-square test were used in the study.Results:? WSB2 was detected in human melanoma cell lines such as A375,G361.and A2058? WSB2-shRNA lentiviral interference vector can infect A375 and G361 cells? WSB2-shRNA lentiviral interference vector can reduce the expression of WSB2 in A375 and G361 cells? Knockdown of WSB2 in melanoma cell lines A375 and G361 inhibits melanoma cell proliferation.? Knockdown of WSB2 in melanoma cell lines A375 and G361 inhibits colony formation of melanoma cell.? Knockdown of WSB2 in melanoma cell lines A375 and G361 can cause G1/S phase arrest in cell cycle.? Knockdown of WSB2 in melanoma cell lines A375 and G361 promotes early apoptosis and late apoptosis of melanoma cells;? Knockdown of WSB2 in melanoma cell lines A375 and G361 inhibits melanoma cell migration;? Sh-WSB2 A375 cells were transplanted into nude mice,whose tumor growth was inhibited.Conclusion:WSB2 probably promotes the proliferation,migration,cell cycle progression and inhibits apoptosis of melanoma cells.WSB2 may promote the growth of melanoma in vivo.Part ?:WSB2 activates Wnt/?-Catenin and MAPK signaling pathway to promote proliferation,migration and inhibit apoptosis of melanoma cellsObjective:In the previous study,it was found that WSB2 probably promotes malignant procession of melanoma.Molecular mechanism of WSB2 promoting tumor cell proliferation,migration and inhibition of apoptosis is explored by westernblotting.It may provide a reliable scientific basis for WSB2 to become a new target for melanoma treatment.Methods:First,after inhibiting the expression of WSB2,proteins in Wnt/?-Catenin signaling pathway,MAPK signaling pathway and apoptotic pathway were detected by western blot.Then,using the recovery experiment,the relationship between the related signaling pathway protein molecules and WSB2 was verified.One-way anova analysis,t-test,and chi-square test were used in the study.Results:? Knockdown of WSB2 in A375 cells,cyclinB1,cyclinD1,p16,p27,p53 expression increased,CDK2,CDK4,CDK6,cyclinD3,p-Rb expression decreased.? Knockdown of WSB2 in A375 cells,c-Myc,?-catenin,caspase-3,Bcl-2,Bad,Cytotic C decreased,Bax,p53,Cleaved caspase-3,caspase-7 expression increased.? Knockdown of WSB2 in A375 cells,the expression of Ras,b-Raf,MEK1,MEK2,JNK,c-Jun,c-Myc,PI3K,STAT3 decreased.? The expression of WSB2 and cell viability in LV-shWSB2 G361/A375 cell decreased significantly compared to LV-scramble group.After given SKL2001(?-catenin agonist),cell viability was restored.Conclusion:The mechanism of WSB2 inhibition of melanoma cell apoptosis may attribute to down-regulation of WSB2 expression,effective inhibition of c-Myc and Bcl2,up-regulation of Bax,leading to a decrease in Bcl2/Bax ratio.Abnormal activation of Wnt/?-catenin signaling pathway and MAPK signaling pathway may be a potential mechanism for WSB2 to promote melanoma proliferation.WSB2 is expected to be a new therapeutic target for melanoma patients,but more experiments are needed.