The Role Of Pin1 In Melanoma Stem Cells

Objective: Explore the role of Pin1 in melanoma stem cells and evaluate the potential of Pin1 targeted therapy,to provide experimental basis and open up new ideas for the treatment of melanoma.Methods: 1.Package lentivirus expressing PIN1 sh RNA,infect melanoma cell lines and create stable Pin1 gene knockdown cells with drug screening.Explore effects of Pin1 gene knockdown on the expression of stem cell marker in melanoma cell lines by Western Blot and flow cytometry.2.Use soft agar assay and melanoma stem cell sphere formation assay to investigate the influence of Pin1 gene knockdown on melanoma cells clone proliferation and sphere formation.3.Melanoma cells with or without stable Pin1 gene knockdown were subcutaneously injected into the left and right flanks of the same nude mice,respectively,to explore the capacity of melanoma cells tumorigenicity in nude mice vivo after Pin1 gene knockdown.4.Melanoma cells with stable Pin1 gene knockdown were serially diluted and injected into nude mice subcutaneously to investigate tumorigenicity of melanoma stem cells in nude mice.5.The nude mice were randomly divided into experimental group and control group.In the experimental group,the mice tail vein were injected with 2*10^6 cells with Pin1 gene knockdown,while the control group was injected with the same number of control cells,to test the effect of Pin1 knockdown on the capacity of melanoma cells lung metastases.6.Inject melanoma cells into nude mice subcutaneously to grow tumors,and the nude mice were randomly divided into 2 groups treated with Pin1 inhibitor ATRA alone or 4 groups treated ATRA combined with Vemurafenib,to evaluate the therapeutic effect of Pin1 inhibitor ATRA treated alone or ATRA combinedwith Vemurafenib on melanoma growth in vivo.7.The human clinical specimens and melanoma tissue microarray were used to detect the expression level of Pin1 by immunohistochemical techniques.Results: 1.Western Blot showed that when Pin1 protein was down regulated,NANOG protein associated with the stemness of melanoma stem cells was also down regulated,while MITF protein associated with differentiation of cancer stem cells was upregulated.2.Flow cytometry results indicated that Pin1 knockdown leads to downregulation of stem cell markers CD271 and ALDH1 as compared to control.3.Soft agar colony formation assay illustrated that clone proliferation capacity of the melanoma cells were decreased by Pin1 gene knockdown;melanoma stem cell sphere formation assay indicated that the number and volume of melanospheres were reduced in Pin1 knockdown group as compared to control.4.Pin1 gene knockdown inhibited the xenograft tumor growth under nude mice skin and the lung metastasis in nude mice.5.Melanoma cells with stable Pin1 gene knockdown were serially diluted and injected into nude mice subcutaneously.It was manifested that none tumor can be formed with 1*10^3,1*10^4,1*10^5cells.6.Human melanoma cells xenografted into nude mice subcutaneously were treated with ATRA alone.The resultes revealed that compared with the control group,Pin1 inhibitor ATRA can inhibit the growth of tumor in vivo.7.By immunohistochemical techniques to detecte Pin1 expression level in human clinical specimens and melanoma tissue microarray,22 among 156 samples have high expression(+++),43 cases medium expression(++),46 cases low expression(+),and 45 cases none expression(-).Conclusions: Our research results demonstrated that there are 14.1% tumor tissue of melanoma patients expressing high level of of Pin1.With Pin1 gene knockdown,melanoma cells clone proliferation was reducted,the melanoma markers associatedwith stemness or differentiation was dysregulated,melanoma stem cell markesr and melanospheres formation capacity were also downregulationed.Both human melanoma cells tumor growth and the capacity of lung metastasis in nude mice were restrained by Pin1 knockdown of melanoma cells.the human melanoma cells subcutaneous tumor growth in nude mice was inhibited by Pin1 inhibitor ATRA.Overall,we believe that Pin1 promote melanoma development through regulating melanoma stem cells,and that the application of Pin1 inhibitors may provide a new way for the clinical treatment of melanoma.