| Malignant melanoma?MM?mostly occurs on the skin and mucous membrane.It is malignant tumor that originates from embryonic neural crest.It is easy to metastasize and has a poor prognosis.The incidence rate is 1%to 3%of all the malignant tumors.However,the incidence of malignant melanoma has increased over the past decades.Traditional therapies like surgery,radiotherapy and chemotherapy have been the main methods for the treatment of malignant melanoma,but for advanced patients,surgical treatment has no survival benefit,and it is not sensitive to radiotherapy and chemotherapy.In recent years,with the discovery and in-depth study of melanoma-associated mutant genes,molecule-targeted drugs have brought breakthroughs in the treatment.Therefore,exploring more effective targets has become the top priority.Epidermal growth factor receptor?EGFR?is a protein with a molecular weight of 170 kDa,and is an important substance involved in proliferation,migration and survival of normal cells and abnormal cells.Epidermal growth factor?EGF?is its ligand.When EGFR is stimulated,it can bind with EGF,resulting in configurational changes,forming homo-dimer or hetero-dimer,making its receptor tyrosine protein kinase autophosphorylates and activates its downstream pathway.Among them are Ras/Raf/MAPK and PI3K/AKT pathways.At present,studies have shown that overexpression of EGFR can be found in many human tumors,and the expression level of EGFR can be related to poor therapeutic effect,rapid disease progression,and low survival rate.Caveolin-1?Cav-1?is a membrane intrinsic protein with a molecular weight of 21-24 kDa.It is an important structural protein of invaginated membrane vesicles?caveolae?.Cav-1 is composed of 178 amino acid residues,and the hydrophobic residues?102-134?located in the peptide chain form a special hairpin-like structure on the membrane structure,dividing the Cav-1 protein into two regions,N-terminus and C-terminus.The N-terminus is the main functional region,which relates to EGFR and regulates the activity of EGFR tyrosine kinase.Studies have shown that Cav-1 exerts anti-tumor effects in cell lines such as bladder cancer.However,studies on the role of Cav-1 in the regulation of EGFR in malignant melanoma cells have not been reported.According to preliminary experimental results,the expression of Cav-1in malignant melanoma UACC647 cells was significantly higher than that in M2 cells,whereas the expression of Cav-1 in M2 cells was less.Therefore,in this study,UCCC647 cells with high expression of Cav-1 and M2 cells with low expression of Cav-1 were studied.The expression level of Cav-1 in the two groups of cells was changed through plasmid transfection technology,and then the effect of tumor cell proliferation,migration,and invasion was observed after that change.In addition further explore its possible molecular mechanisms.To provide a new theoretical basis for the treatment of malignant melanoma.Methods:1 Cell transfectionThe UACC647 cells with high expression of Cav-1 were selected and transfected with Ctrl shRNA and Cav-1 shRNA plasmids respectively.The puromycin resistance was screened to construct stable transfected UACC647?Ctrl?and UACC647?KD?cells.In addition,M2 cells with low expression of Cav-1 were selected,and pcDNA3.1 and pcDNA3.1/Cav-1 plasmids were transfected respectively.G418 was used for resistance screening,and stably transfected M2?pcDNA3.1?and M2?pcDNA3.1/Cav-1?cells were constructed.Western blot verified the expression of Cav-1.2 MTS test cell proliferationThe cells were seeded in a 96-well plate,and after the cells were attached to the wall of flask,different concentrations of EGF solution were added to stimulate the cells.After a certain period of time,the absorbance?OD?of each well was measured.3 cell scratch test to detect cell migrationThe cells were seeded in a 24-well plate.After forming a single cell layer,the cells were scratched with a pipette tip to observe the migration of cells with or without EGF stimulation.The cells were photographed at different time points until healing.4 Transwell chamber migration assay to detect cell migrationThe cells were seeded in Transwell chambers and incubated in a 37°C,5%CO2 incubator for 24 hours.Observe and count the number of cells that pass through the membrane.5 Western blot detection of protein expression levels of UACC647?Ctrl?,UACC647?KD?cells,M2?pcDNA3.1?and M2?Cav-1?cells cultured in medium and serum-free medium at a final concentration of 20 nM EGF for 0,5,10 and 30min,respectively.The total protein was extracted from each group.Protein samples were sampled in equal volumes,electrophoresed,transferred to membranes,and incubated with antibodies to detect the expression levels of EGFR,pY-EGFR,Akt,p-Akt,Erk,p-Erk,Cav-1,and?-actin in UACC647?Ctrl?,UACC647?KD?,M2?pcDNA3.1?and M2?Cav-1?cells,respectively.The expression levels were measured by measuring gray values.The relative amounts of phosphorylated EGFR,Akt,and Erk account for the total EGFR,Akt,and Erk were calculated Value respectively.Results:1 Expression level of Cav-1 protein in stably transfected cell linesWestern blot showed that the relative gray levels of Cav-1 protein in UACC647?Ctrl?and UACC647?KD?cells were 0.84±0.03 and 0.24±0.01,respectively.Compared with untransfected group and empty plasmid transfected group,the expression of Cav-1 protein in Cav-1 shRNA plasmid transfection group was reduced by more than 70%,the difference was significant?P<0.01?.The relative gray values of Cav-1 expression in M2?pcDNA3.1?and M2?Cav-1?cells were 0.83±0.08 and 2.01±0.21,respectively.Compared with the untransfected group and the blank control group,the expression of Cav-1 protein in the Cav-1 plasmid transfection group was significantly higher,and the difference was significant?P<0.01?.2 Effect of Cav-1 on proliferation of malignant melanoma cellsMTS assay was used to detect changes in the proliferation of UACC647cells after knockdown of Cav-1 expression.Under the stimulation of EGF with final concentrations of 4 nM,20 nM,and 100 nM,the activity of UACC647?KD?cells with low expression of Cav-1 was increased compared with UACC647?Ctrl?cells that normally expressed Cav-1?0.78±0.02 vs0.88±0.04,0.86±0.01 vs 1.03±0.03,and 0.98±0.03 vs 1.15±0.03?.The differences were statistically significant?P<0.05?.After overexpression of Cav-1,M2 cells were stimulated with EGF with final concentrations of 0 nM,4 nM,20 nM,and 100 nM,respectively,compared with M2?pcDNA3.1?cells that normally express Cav-1,the cell viability of M2?Cav-1?was decreased?0.92±0.03 vs.0.63±0.05,1.03±0.11 vs.0.73±0.06,1.10±0.10 vs.0.80±0.02,1.22±0.09 vs.0.89±0.03?,and the differences were significant.?P<0.01?.3 Effect of Cav-1 on migration of malignant melanoma cellsThe wound-healing assay was used to detect the change of migration ability of UACC647 cells after knockdown of Cav-1 expression level.The cells were photographed at the same scratch position in each group of cells to observe the healing condition of the cells.The initial width of the scratch was set to 1,and the relative width of scratches at each time point was calculated.Under the condition of no EGF stimulation,the UACC647?KD?cells with low expression of Cav-1 showed faster wound healing,and the relative width of scratches was smaller than that of normal UACC647?Ctrl?cells expressing Cav-1?0.72±0.02 vs.0.87±0.02,0.67±0.01 vs.0.81±0.03,0.46±0.01 vs.0.76±0.01?,the differences were significant?P<0.01?.In the presence of EGF stimulation,UACC647?KD?cells also recovered faster at each time point,and the relative width of scratches was significantly lower than that of UACC647?Ctrl?cells?0.57±0.03 vs.0.75±0.02,0.28±0.03 vs.0.68±0.02,0.09±0.01 vs.0.49±0.01?,the differences were significant?P<0.01?.The same method was used to detect the migration ability of M2?Cav-1?cells.In the absence of EGF stimulation,the wound healing rate of M2?Cav-1?cells was slower at each time point,and the relative width of scratch healing was greater than that of M2?pcDNA3.1?cells?0.92±0.03 vs.0.72±0.02,0.81±0.05 vs.0.57±0.03,0.65±0.01 vs.0.34±0.04?,the differences were significant?P<0.01?.In the presence of EGF stimulation,M2?Cav-1?cells also showed slower healing at different time points than M2?pcDNA3.1?cells,and the relative width of scratches was significantly larger?0.82±0.03 vs.0.63±0.03,0.73±0.02 vs.0.42±0.02,0.46±0.03 vs.0.00±0.00?,the differences were significant?P<0.01?.4 Effect of Cav-1 on invasion of malignant melanoma cellsTranswell assay was used to detect the change of the invasive ability of UACC647 cells after knockdown of Cav-1 expression.Compared with UCCC647?Ctrl?cells that normally expressed Cav-1,the invasion ability of UACC647?KD?cells with low expression of Cav-1 was increased,and the relative value of cells passing through Transwell chamber membrane was significantly higher?1±0.00 vs.2.37±0.09?,the difference was significant?P<0.01?.The same method was used to detect the changes of invasive ability in M2 cells which overexpressed Cav-1.M2?Cav-1?cells were less invasive than M2?pcDNA3.1?cells.The relative value of cells passing through the Transwell chamber membrane was significantly reduced?1±0.00 vs.0.59±0.08?,with a significant difference?P<0.01?.5 Effect of Cav-1 on protein expression of MAPK and PI3K signaling pathwayUACC647?Ctrl?cells that normally expressed Cav-1 and Cav-1low-expressing UACC647?KD?cells were selected.Western blot was used to detect the levels of phosphorylated EGFR,Erk,and Akt after 0,5,10,and30 minutes of EGF stimulation.Total protein of UACC647?Ctrl?and UACC647?KD?cells were extracted,and Cav-1 antibody test to confirmed that the expression of Cav-1 in UACC647?KD?cells was significantly reduced,providing a basis for subsequent analysis of silencing Cav-1affecting EGFR signaling pathway.UACC647?Ctrl?and UACC647?KD?cell total proteins were detected with anti-phospho EGFR.Statistical analysis showed that the relative phosphorylation of EGFR in UACC647?KD?cells at various time points?5,10,and 30 minutes?was significantly higher than that of UACC647?Ctrl?cells under EGF stimulation conditions?4.41±0.11 vs.1.80±0.20,1.35±0.07 vs.0.78±0.09,1.19±0.05 vs 0.51±0.14?,the differences were significant?P<0.01?.UACC647?Ctrl?and UACC647?KD?cell total proteins were detected with anti-phosphorylated Erk.Statistical analysis showed that under EGF stimulation conditions,the relative value of phosphorylated Erk at each time point?5,10,and 30 minutes?of UACC647?KD?cells was significantly higher than that of UACC647?Ctrl?cells?0.89±0.05 vs.0.37±0.03,0.95±0.02 vs.0.65±0.03,0.90±0.04 vs.0.72±0.02?,the differences were significant?P<0.05?.UACC647?Ctrl?and UACC647?KD?cell total proteins were detected with anti-phospho-Akt antibodies.Statistical analysis showed that the relative phosphorylation of Akt in UACC647?KD?cells at various time points?5,10,30 minutes?was significantly higher than that of UACC647?Ctrl?cells?0.69±0.02 vs.0.38±0.06,0.78±0.05 vs.0.38±0.06,1.08±0.05 vs.0.68±0.05?under EGF stimulation conditions,the differences were significant?P<0.01?.It is known that down-regulating Cav-1 in cells promotes EGF-induced activation of EGFR and activates downstream MAPK/ERK and PI3K/AKT pathways.The same method was used to detect the phosphorylation of EGFR,Erk and Akt levels in M2?pcDNA3.1?and M2?Cav-1?cells after EGF.In the EGF-stimulated condition,the relative phosphorylation of EGFR in M2?Cav-1?cells at each time point?5,10,30 min?was significantly lower than that of M2?pcDNA3.1?cells?1.41±0.10 vs.2.60±0.28,0.36±0.04 vs.1.83±0.06,0.21±0.07 vs.0.67±0.08?,the differences were significant?P<0.01?;the relative value of phosphorylated Erk was significantly lower than that of M2?pcDNA3.1?cells?0.72±0.05 vs.1.15±0.04,0.88±0.07 vs.1.47±0.09,0.68±0.08 vs.1.36±0.03?,the differences were statistically significant?P<0.01?;the relative value of phosphorylated Akt was significantly lower than that of M2?pcDNA3.1?cells?0.56±0.02 vs.1.16±0.05,0.40±0.08 vs.1.93±0.07,0.37±0.06 vs.1.26±0.09?,the differences were statistically significant?P<0.01?.It is known that when Cav-1 is over-expressed in cells,EGF-induced EGFR activation may be decreased,and downstream MAPK/ERK and PI3K/Akt pathways may be inhibited.In summary,Cav-1 can inhibit downstream MAPK/ERK and PI3K/AKT signaling pathways by regulating the activation of EGFR.Conclusion:1 Knockdown of Cav-1 expression can promote the proliferation,migration and invasion of human malignant melanoma cells;2 Overexpression of Cav-1 inhibited the proliferation,migration and invasion of human malignant melanoma cells.3 Cav-1 can inhibit the growth of malignant melanoma cells by regulating the activation of EGFR and inhibiting downstream MAPK/ERK and PI3K/AKT signaling pathways. |
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