An Analysis Of Genes VAMP7、VAMP8 In Breast Cancer Based On Immunohistochemistry And Cell Experiments In Vitro

Objective:（1）By collecting specimens of breast cancer（BC）cases,conducting immunohistochemical experiments related VAMP7,VAMP8 genes protein expression in BC tissue,in order to analysis the relationship between target genes and clinical pathological parameters,to reveal its role in BC based on clinical data support;（2）Stable transfection expressing BC cell lines with knockdown VAMP7 and VAMP8 genes and relevant cell function experiments were conducted to analyze the effect of VAMP7,VAMP8 genes on BC cell lines;（3）Through collection and integration of multiple platforms BC microarray chips and BC patients’clinical information,preliminarily analyze VAMP7,VAMP8 gene in BC and its mRNA and protein levels differences in expression,molecular mechanism,diagnosis and prognosis value,specific the role of VAMP7,VAMP8 gene in the BC.Materials and methods:1.Immunohistochemistry was used to detect the VAMP7,VAMP8 protein expression levels in paraffin specimens of mammary tissue of 100 BC patients and 100 non-cancer patients,and to explore the differential expression of protein expression in BC and the relationship between these protein expressions and the clinicopathological characteristics of BC patients;2.MCF-7,MDA-MB-231 and SK-BR-3 BC cell lines were stably expressed using lentivirus transfecting and purinamycin selecting after knockdown of VAMP7 or VAMP8,and functional experiments such as cell migration,cell proliferation and cell invasion were conducted to verify whether knockdown of VAMP7 or VAMP8 genes and reducing the expression of mRNA or protein can inhibit the biological behavior of BC cells;3.through collecting Gene Expression Omnibus（GEO）and the Cancer Genome Atlas（TCGA）associated with BC in microarray chip data,extracting VAMP7 and VAMP8messages in 60 sets of microarray chip of gene expression in cancer and non-cancer value,through standardized processing,mRNA expression levels of BC and non-cancerous tissue were contrasted,VAMP7 or VAMP8 diagnosis efficiency and its relationship between clinical parameters were analyzed.Results:1.Immunohistochemical results suggested that the protein levels of VAMP7 and VAMP8 were higher in BC than in non-cancer tissues,and the statistical analysis was significant(P_VAMP7<0.001;P_VAMP8<0.001),and VAMP7is related to the onset age of BC patients（P=0.021）,and VAMP8 is related to tumor size（P=0.019）,and there are no obvious correlations with other clinical-pathological parameters;2.build stable BC cell lines after RT-q PCR experiments confirmed that the VAMP7 mRNA level of LV-VAMP7 group,the VAMP8 mRNA level of LV-VAMP8 group were lower than that of blank group and negative control group（P<0.05）,indicating that successfully build low expressed VAMP7 or VAMP8 cell lines.Cell function experiments suggest that VAMP7 or VAMP8 low expression weakened cell invasion and migration ability（P<0.05）,VAMP8 gene knock down reduce three types of BC cell line proliferation,VAMP7 only an obvious effect in inhibiting MDA-MB-231;3.microarray chips into 4172 BC samples,and 890 cases of non-BC samples,60set chips were divided into 20 platforms,which can extract 16 groups of VAMP7 data and 20 groups of VAMP8 data.There are 11 in 16 chip sets prompt the VAMP7 expression higher in BC tissue than non-cancerous tissue（SMD=0.33,95%CI:0.02,0.63）,the area under the curves（AUC）of ROC is 0.7479,the sensitivity is 0.88,the specificity is 0.33,positive likelihood ratio is 1.46,negative likelihood ratio is 0.44.There are 16 groups prompt the VAMP8expression higher in BC tissue than non-cancerous tissue and SMD is 0.61,95%CI（0.26,0.95）,the AUC of sROC is 0.7662,sensitivity is 0.86,specificity is 0.46,positive likelihood ratio is 1.84 and negative likelihood ratio is 0.37.Conclusion:1.VAMP7 and VAMP8 protein express high in BC tissue than non-cancer tissue,VAMP7 high expression related to onset age of BC cases,and VAMP8 high expression related to tumor size of BC cases;2.VAMP7 or VAMP8 low expression weakened BC cells invasion and migration ability,VAMP8 express low reduce three types of BC cell proliferation,VAMP7 express low only inhibit MDA-MB-231 cell line;3.VAMP7,VAMP8 mRNA level differentially expressed in BC and adjacent tissues,while they have the ability to diagnose BC,its specificity is not good enough.