The Role And Molecular Mechanism Of CYLD In Melanoma

Melanoma is one of the malignant tumors that produced by the malignant transformation of melanocytes,which mostly occurs in the skin.Malignant melanoma of the skin is mostly formed by the development of normal moles and pigmented spots.Malignant melanoma of the skin grows fast,has strong metastasis and has a high mortality rate.It has great significance for exploring its pathogenesis to the elucidation and treatment of melanoma.Cylindromatosis(CYLD)is a tumor suppressor gene,and its mutation is closely related to the occurrence of human familial cylinder tumors.CYLD is a deubiquitinating enzyme that binds to microtubules.It is widely distributed in human cells and can regulate the level of protein ubiquitination.CYLD plays a role in various biological processes such as inflammation,cell proliferation and migration.We analyzed the expression of CYLD in cutaneous melanoma through the sample data of cutaneous melanoma patients in the tcga database.It is found that it is lowly expressed in patients and closely related to survival time.It is suggesting that CYLD plays an important role in the development of cutaneous malignant melanoma.However,the mechanism is not yet fully clear about the role of CYLD and its molecules in cutaneous melanoma.Therefore,this paper uses human malignant cutaneous melanoma cell lines to explore the role of CYLD in the advancement of tumor cell proliferation and its molecular mechanism.First,we extracted the sample data of cutaneous melanoma patients in the TCGA database,and analyzed the relationship between the expression of CYLD and the survival time of cutaneous melanoma patients.Further,protein expression verification was performed on human skin melanocytes and six human malignant skin melanoma cell lines,from which two melanoma cell lines were selected for functional studies.Construction of CYLD OE,CYLD-sh RNA,OE-NC and sh RNA-NC stably transformed melanoma A375 cell line and C8161 cell line by using lentivirus infection method.Next,explore the effect of CYLD on melanoma cell proliferation and migration by Ed U and scratch injury experiments.In order to explore the molecular mechanism of CYLD regulating tumor cell proliferation and migration,this study screened CYLD interacting proteins through immunoprecipitation-mass spectrometry,and further used immunoprecipitation experiments to verify the interacting proteins.We validate the level of ubiquitination modification of interacting proteins through ubiquitination modification analysis experiments and preliminary study on the mechanism of CYLD inhibiting proliferation and migration of cutaneous melanoma cells.The experimental results are as follows: 1.CYLD is lowly expressed in human cutaneous melanoma tissues and cutaneous melanoma cell lines.It was found that CYLD protein was lowly expressed in melanoma patients,and the survival time of patients with low expression of CYLD was shorter by analyzing the SKCM sample data of the TCGA database.CYLD expression levels of human normal skin melanocytes(PIG1)and six human malignant skin melanoma cells(SK-MEL-1,SK-MEL-2,SK-MEL-28,C8161,A375,WM115)were detected by immunoblotting experiments.The results showed that compared with PIG1 cells,six human melanoma cells all under-expressed CYLD protein,suggesting that CYLD protein expression is closely related to the development of melanoma.2.CYLD significantly inhibits the proliferation and migration of melanoma cells.We constructed CYLD OE,OE-NC,CYLD-sh RNA,sh RNA-NC of A375 and C8161 stable transfected cell lines using lentivirus.Analysis of cell proliferation and migration using Ed U and scratch damage experiments.Ed U experiment results show that CYLD overexpression has a very significant inhibitory effect on melanoma cell proliferation(p?0.01)and knockdown significantly promotes cell proliferation(p?0.01);cell scratch experiments showed that overexpression of CYLD significantly inhibited the migration of melanoma cells(p?0.01),while knocking down significantly promoted the cell migration ability(p?0.01).The experimental results show that CYLD significantly inhibits the proliferation and migration of melanoma cells.3.Molecular Mechanism of CYLD Inhibiting Proliferation and Migration of Melanoma Cells.In order to explore the molecular mechanism of CYLD inhibiting melanoma cell proliferation and migration,this study overexpressed Flag-CYLD in human malignant melanoma cell lines,using immunoprecipitation-mass spectrometry to identify proteins interacting with CYLD.After screening,it was found that NPM may be Interact with CYLD.Further,we successfully constructed the Flag-NPM eukaryotic expression vector using molecular cloning technology and overexpressing Flag-NPM and GFP-CYLD in 293 T cells to explore the interaction between the two proteins by using immunoprecipitation technology.The results show that there is an interaction between CYLD and NPM.In order to investigate whether CYLD regulates the level of ubiquitination of NPM,overexpression of Flag-NPM,GFP-CYLD and Myc-Ub expression vectors in 293 T cells.Immunoprecipitation experiments showed that CYLD can reduce the ubiquitination level of NPM,indicating that NPM is the deubiquitination substrate of CYLD.Further,we analyzed PD-L1 protein in human normal melanocytes(PIG1)and six human malignant skin melanoma cells(SK-MEL-1,SK-MEL-2,SK-MEL-28,C8161,A375,WM115).The results show that PD-L1 is highly expressed in malignant melanoma cells.Further analysis showed that CYLD can regulate the expression of PD-L1.High expression of CYLD can inhibit PD-L1 expression,while knocking down CYLD is the oppositeCombining the relevant literature and the results of this experiment,We believe that CYLD is lowly expressed in cutaneous malignant melanoma.CYLD interacts with NPM to regulate the level of NPM ubiquitination and affect the expression of PD-L1,which in turn affects cell proliferation and migration.These data provide data support for exploring the role and mechanism of CYLD in cutaneous malignant melanoma.