NCAM Affects The Migration Of Human Malignant Melanoma Cells Via Src/Akt Signal-mediated Cofilin Activity

BackgroundMelanoma is a highly metastatic malignant tumor derived from neuromelanocytes,which can occur in various parts of the patient's body.Although there are many methods to treat tumors,the prognosis of metastatic melanoma patients is still not optimistic.Therefore,it is an urgent problem to further study the metastatic development mechanism of malignant melanoma and find the effective target to prevent and treat melanoma metastasis.Neural cell adhesion molecule(NCAM),a member of immunoglobulin superfamily,is highly expressed in the nervous system and mainly involved in regulating the adhesion,migration,differentiation and synaptic formation of nerve cells.Recent studies have found that NCAM is also involved in the invasion,development and metastasis of tumor cells.Our previous studies on bone marrow mesenchymal stem cells showed that knockout of NCAM gene could inhibit the formation of directional pseudopodia and thus affect the migration ability of cells.However,the role and mechanism of NCAM in human malignant melanoma are still unclear.The purpose of this study is to evaluate the effects of NCAM on the migration of human malignant melanoma A375 and M102 cells and the possible molecular mechanisms.Objective1.Investigate the effects of NCAM on the migration of human malignant melanoma cells A375 and M102;2.Explore the molecular mechanism of NCAM gene affecting human malignant melanoma cell migration.Methods1.Screening of stable human malignant melanoma cellHuman malignant melanoma A375 and M102 cells were infected with lentivirus carrying shNCAM plasmid to obtain stable shNCAM-expressing cell lines.After 96 h,the cells were further cultured in a medium containing puromycin at the optimal screening concentration to obtain two stable cell lines of human malignant melanoma by NCAM gene silencing.2.Examining the effect of NCAM on the migration of human malignant melanoma cellsAfter NCAM gene silencing,the migration of human melanoma cells was assessed by scratch healing assay and transwell migration assay.3.Examining the effects of NCAM on actin cytoskeleton rearrangement of human malignant melanoma cellsThe immunofluorescence assay and western blot were performed to analysis the effects of NCAM gene silencing on the rearrangement of actin cytoskeleton and the activity of actin binding protein cofilin in human malignant melanoma cells.4.The mechanism of NCAM affecting actin cytoskeleton rearrangement in human malignant melanoma cellsFirstly,we searched for the molecules that were located in the downstream of NCAM and related to cytoskeleton rearrangement by reading the literature,and finally the Src/Akt signaling pathway was targeted.Then the influence of NCAM gene silencing on Src and Akt phosphorylation levels was detected by western blot.Further,Src inhibitor PP2 and Akt inhibitor LY294002 were added to normal melanoma cells to detect the effect of Src/Akt signaling pathway on cofilin activity.Results1.We successfully obtained two stable transfected cell lines with NCAM gene silencing,and the obtained stable transfected cell lines were used for subsequentexperiments;2.NCAM gene silencing inhibited the migration of human malignant melanoma A375 and M102 cells;3.NCAM gene silencing inhibited the phosphorylation of cofilin in human malignant melanoma cells A375 and M102,which in turn affects actin cytoskeleton rearrangement;4.NCAM gene silencing inhibited Src and Akt phosphorylation in human malignant melanoma cells A375 and M102;The level of phosphorylated cofilin was reduced when Akt and Src inhibitors were added to A375 and M102 cells,respectively.Conclusion1.NCAM regulated the migration of human malignant melanoma cells A375 and M102;2.NCAM may affect the phosphorylation of cofilin through Src/Akt signaling pathway,which regulated the rearrangement of actin cytoskeleton in melanoma cells.