PAI-1 Promotes Metastasis Of Human Cutaneous Melanoma

BackgroundThe human cutaneous melanoma is of skin malignant tumor,Clinical characteristics of human cutaneous melanoma include no particular symptoms in the early stage,high rate of disease recurrence and easily metastasize to distinct sites.Growing evidence reveals that epithelial mesenchymal transition(EMT)is closely related to the malignant tumor invasion and metastasis.Therefore,EMT is a new strategy for the treatment of cutaneous malignant melanoma on invasion and metastasis.EMT was regulated by intrinsic cellular pathways as well as extrinsic signals generated by the tumor microenvironment.Plasminogen activator inhibitor-1(PAI-1)play an important role on EMT in tumor microenvironment.However,the effect of PAI-1 on human cutaneous melanoma remains to be detailed explored.The thesis discusses these.Objectives1.Detect the expression of PAI-1 in human cutaneous melanoma.2.Define the function of PAI-1 in the metastasis of human cutaneous melanoma.3.Clarify the mechanisms of PAI-1 on human cutaneous melanoma.Methods1.Detect the expression of PAI-1 in human cutaneous melanoma.(1)Using the immunohistochemistry to analyze the expression of PAI-1 in human fresh cutaneous melanoma tissue.(2)Verifying the expression of PAI-1 in human fresh cutaneous melanoma tissue by immunofluorescence assay.2.Evaluate the function of PAI-1 in the metastasis of human cutaneous melanoma.(1)Studying the function of PAI-1 in the metastasis of human cutaneous melanoma cell line A375 and SK-MEL-31 by scratch,invasion and metastasis of experiments.(2)Studying the function of overexpressed PAI-1 in the metastasis of human cutaneous melanoma cell line A375 and SK-MEL-31 by scratch,invasion and metastasis of experiments.(3)Studying the function of PAI-1 in the metastasis of human cutaneous melanoma cell line A375 by transfecting A375 with PAI-1-expressing Lentivirus and nude mouse tumor-metastasis model.3.Investigate the mechanisms of PAI-1 on human cutaneous melanoma cell(1)Verifying the NF-?B signaling pathway in PAI-1 on human cutaneous melanoma cell by western-blot,immunofluorescence.(2).Verifying the NF-?B signaling pathway in PAI-1 on human cutaneous melanoma cell by antibody inhibitor assay.Results1.Expression of PAI-1 on the human cutaneous melanomaImmunohistochemistry and immunofluorescence assay revealed that the percentage of PAI-1 expressing cells in the human fresh cutaneous melanoma tissue.The PAI-1 positive-staining cells were located mainly in the stroma and tumor.Further confirm that the majority of PAI-1 positive-staining cells were CD68+ cells.2.PAI-1 promotes human cutaneous melanoma cell metastasisBy scratch,invasion and metastasis of experiments,the number of cells significantly increased by PAI-1 stimulation in vitro.By scratch,invasion and metastasis of experiments,the number of cells significantly increased by overexpressed-PAI-1cells in vitro.To further evaluate the role of PAI-1 on human cutaneous melanoma cell metastasis in vivo,we get ectopic expression of PAI-1 in human cutaneous melanoma cell using lentivirus-mediated transduction to mimic PAI-1,human cutaneous melanoma cell A375 transfected with PAI-1 showed greater metastasis capacity in mice.3.NF-?B signaling pathways are involved in PAI-1-enhanced metastasis of human cutaneous melanoma.After stimulated by PAI-1(24h),the expression of E-cadherin is decreseaed and Vimentin increased.Western-blot and immunofluorescence assay showed that PAI-1 stimulation was associated with a significant increase in the level of p65 in the nuclear extracts at 5min and 15 min.To further verify the role of NF-?B signaling in PAI-1-enhanced metastasis,we used NF-?B inhibitor PDTC to block its activation during the treatment of PAI-1.As expected,we found that the increased number of cells caused by PAI-1 treatment could be abolished by PDTC treatment.ConclusionPAI-1-producing cells(CD68+ cells)were components of tumor microenvironment in human cutaneous melanoma.PAI-1 could promote metastasis in vitro and enhance metastasis potential in xenograft mice.And these effects were mediated by NF-?B signaling.