| BackgroundMelanoma,a skin cancer that arises from the cancerated neural crest melanocytes,is mostly found in skin,nasal cavity and eyes.In its early stages,malignant melanoma can be removed by a surgeon.but once it has progressed to the advanced metastatic stage,it is extremely difficult to cure and does not respond to current therapies.Neural cell adhesion molecule(NCAM),a novel immunoglobulin superfamily type cell adhesion molecule,was first found and described in the central nervous system.It has been reported that NCAM plays an crucial role in tumor progression.So far,the capacity of NCAM in murine melanoma B16F0 cells is still unknown.Objectives 1.To analyze the function of NCAM140/180 in murine melanoma B16F0 cell proliferation,migration and invasion;2.Explore the related mechanism of NCAM140/180 in promoting the metastases of murine melanoma B16F0 cell.Method 1.The effect of NCAM140/180 gene overexpression in murine melanoma B16F0 cells proliferationThe NCAM140/180 adenovirus vectors were infected into B16F0 cells,48 h later,the cells were seeded into 96 well plate,the effect of NCAM140/180 gene overexpression of B16F0 cells was measured by MTT assay.The NCAM140/180 overexpressed B16F0 cells were seeded in 6 well plate,after 4 days later,the clone formation experiment was explored to analyze the effect of NCAM140/180 gene overexpression on cell clone formation ability of B16F0 cells.2.The effect of NCAM140/180 gene overexpression in murine melanoma B16F0 cell proliferation,migration and invasion.The NCAM140/180 adenovirus vectors were infected into B16F0 cells,48 h later,serum starvation for 12 h,a 10 ?L pipette tip was used to make a wound scratch.The wound area was photographed by a microscope at various time points.According to measuring the changed wound area,the ability of migration was calculated.Transwell migration and invasion assay was proceed at the same time,to further evaluate the Changes in cell migration ability.3.The related mechanism of NCAM140/180 in promoting the metastases of murine melanoma B16F0 cell.The NCAM140/180 adenovirus vectors were infected into B16F0 cells,48 h later extract total protein,western blot analyze the related signaling molecular FGFR?AKT?CREB and c-Jun.A PepTag? Non-Radioactive cAMP-Dependent Protein Kinase Assay was carried out to detected the activity of the PKA.However,the B16F0 cells were treated with specific inhibitor,the capacity of B16F0 cell proliferation,migration and invasion was remarkable inhibited,It also confirms the function of related signaling in melanoma.Results 1.NCAM140/180 gene overexpression promote murine melanoma B16F0 cell proliferationThe results of MTT assay showed the number of living cells is more NCAM140/180 overexpressed group than control group.Clone formation experiment showed that NCAM140/180 significant promote the ability of B16F0 cell in forming colonies.2.NCAM140/180 gene overexpression promote murine melanoma B16F0 cell migration and invasionWound healing assay and Transwell migration showed that overexpression of NCAM140/180 promote the migration of B16F0 cells.Transwell invasion assay showed that overexpression of NCAM140/180 promote the invasion of B16F0 cells.3.NCAM140/180 promote murine melanoma progression is dependent on FGFR/AKT/PKA signaling passway.Western blotting showed that the degree of the phosphorylated FGFR,AKT,CREB and c-Jun was increased,and the activity of PKA was also increased when NCAM140/180 gene overexpression.Whereas treated the cells with specific inhibitor,the capacity of murine melanoma B16F0 cell proliferation,migration and invasion was also inhibited.ConclusionOur data showed that overexpression of NCAM140/180 can significant boost the murine melanoma B16F0 cell proliferation,migration and invasion by increasing the levels of the phosphorylated FGFR,AKT,PKA,CREB and c-Jun.It is perhaps a novel target to the treatment of melanoma,the related signaling passway may attract attention of researchers as an important target for anti-melanoma drug development. |
PDF Full Text Request