Impact Of Small Interference RNA-induced Silencing Of Adrenomedullin Gene On The Expression Of VEGF,IL-10 In Malignant Melanoma Cells

Objective:Adrenomedullin(ADM)is an endogenous vascular active peptide found in pheochromocytoma organization,the study found that the vast majority of tumor cells can produce ADM,it plays a role in promoting tumor growth,the high expression of ADM in human melanoma tissues affect the biological behavior of melanoma cells.RNA interference technology can be silent to gene,it can explores the relationship between ADM gene with the occurrence and development of melanoma.It has been confirmed that ADMsiRNA can inhibit the proliferation of siRNA cells via G2/M,and induce of the cell apoptosis in vitro,inhibit the cell proliferation,reduce the invasion ability of A375 melanoma cells in vitro.The tumor cells secrete immunosuppressive substances to inhibit immune function,which is the important mechanism of tumor escaping immunosurveillance and plays an important role in promoting tumor generation and development.Whether ADM gene expression is connected with the immune escape deserves further study.VEGF(vascular endothelial growth factor)and IL-10(interleukins 10)are considered immunosuppressive molecules at present,they take part in the cell transition and escaping immunosurveillance by autocrine and paracrine pathways,create favorable micro environment for tumor occurrence and development.It has been certified in experiment that VEGF and IL-10 can be detected in the supernatant of A375 cells culture,but the siRNA silencing adrenomedullin gene can disturb the expression of immunosuppressive molecules of melanoma cell are rare reports.The experiment use small interference RNA(siRNA)-induced silencing of Adrenomedullin(ADM)gene in a malignant melanoma cell line A375,to assess the influence on the expression of immunosuppressive molecules on A375 cells,and investigate the use of adrenomedullin gene in melanoma malignant behavior do,also provides a new idea for immunotherapy and gene therapy of melanoma.Methods:1.Cells:this experiment adopts the cells derived from human skin melanoma cell lines2.Experimental group and the intervention:A375 cells were classified into 3 groups to be transfected with the selected siRNA(test group),non-specific sequences(negative control group),or remain untransfected(blank control group).3.Immunocytochemical staining:the expressions of ADM in each group were detected by Immunocytochemical staining.4.Immunocytochemical staining:the expressions of VEGF and IL-10 in each group were detected by Immunocytochemical staining.5.ELIS A:the expressions of VEGF and IL-10 in each group were detected by ELISA.6.The statistical analysis was performed with SPSS 17.0,The result of Immunocytochemical was analyzed by Wilcoxon rank sum test,staining.using one-way analysis of variance(ANOVA).p<0.05 was considered statistically significant.Result:1.The effect of siRNA transfection:Because of the action of different RNA sequences is transfected(ADM,nonspecific sequences)and untransfected,each group of A375 cells cultured for 24h,then detected by immunocytochemical staining,the expression of ADM is low,but in blank control and negative control group,the positive expression is higher,the results show that siRNA can effectively inhibit the expression of ADM gene.2.The effect of siRNA silencing adrenomedullin gene on the expression of VEGF in melanoma cell:Owing to the action of different RNA sequences is transfected(ADM,nonspecific sequences)and untransfected,each group of A375cells cultured for 24h,then detected by immunocytochemical staining,VEGF positive expression is visible in A375 melanoma cells,positive staining was mainly located in the cytoplasm,membrane and cell membrane.In blank control and negative control group,the positive expression is higher,the low expression was found in ADMsiRNA transfectde group.ELISA method was used to detect the expression of VEGF in the supernatant fluid(pg/ml),because of being transfected by different siRNA sequences,the level of VEGF in A375 melanoma cells is different also,the expression of VEGF in untransfected group is 192.246±17.330,the expression of VEGF in nonspecific transfected group is187.311±14.450,ADMsiRNA transfected group is96.218±,after transfecting of ADMsiRNA,the expression of VEGF in A375 melanoma cells was significantly lower than that of negative control group and blank control group.Treated by analysis of variance,the analysis of variance indicated the difference was significant(F=129.935,p<0.01),there was no significant difference between the negative control group and the blank control group(p=0.518>0.05),the difference between experimental group and the negative control group was significant(p<0.01),compared with the blank control group the difference was also significant(p<0.01),the results show that siRNA silencing adrenomedullin gene can inhibit the expression of VEGF in A375 melanoma cell.3.The effect of siRNA silencing adrenomedullin gene on the expression of IL-10in melanoma cell:Owing to the action of different RNA sequences is transfected(ADM,nonspecific sequences)and untransfected,each group of A375eells cultured for 24h,then detected by immunocytochemical staining,IL-10 positive expression is visible in A375 melanoma cells,positive staining was mainly located in the cytoplasm,membrane and cell membrane.In blank control and negative control group,the positive expression is higher,the low expression was found in ADMsiRNA transfectde group.ELISA method was used to detect the expression of IL-10 in the supernatant fluid(pg/ml),because of being transfected by different siRNA sequences,the level of IL-10in A375 melanoma cells is different also,the expression of IL-10 in untransfected group is108.822±7.550,the expression of IL-10 in nonspecific transfected group is103.250±4.785,ADMsiRNA transfected group is 37.024±5.770,after transfecting of ADMsiRNA,the expression of IL-10 in A375 melanoma cells was significantly lower than that of negative control group and blank control group.Treated by analysis of variance,the analysis of variance indicated the difference was significant(F=380.245,p<0.01),there was no significant difference between the negative control group and the blank control group(p=0.66>0.05),the difference between experimental group and the negative control group was significant(p<0.01),compared with the blank control group the difference was also significant(p<0.01),the results show that siRNA silencing adrenomedullin gene can inhibit the expression of IL-10 in A375 melanoma cell.Conclusion:1.siRNA can effectively inhibit the expression of ADM gene.2.A375 melanoma cells can express immunosuppressive molecules VEGF and IL-10.3.siRNA silencing adrenomedullin gene can inhibit the expression of VEGF and IL-10 in A375 melanoma cell.