Effects Of Lentivirus Mediated-ADAM17 Gene Overexpression On The Proliferation Of Human Breast Cancer MCF-7 Cell

Objectives To observe the effect of ADAM17 gene overexpression on proliferation of human MCF-7 breast cancer cells.Methods Human breast cancer MCF-7 cells were routinely cultured,and divided into 3 groups: blank control group,transfection control group(transfected with lentivirus blank vector)and transfection group(transfected with ADAM17 overexpression lentivirus).The lentiviral vector carried with red fluorescent protein(RFP).After 96 hours of transfection,the transfection efficiency was observed by fluorescence inverted microscope.After successful transfection,the expression levels of ADAM17 mRNA in MCF-7 cells was detected by Real-time PCR,the expression levels of ADAM17 protein in MCF-7 cells was detected by Western blot.The optical density of cells in each group was measured by MTT assay in order to determine the proliferation of cells.The distribution of cell cycle in each group was detected by flow cytometry.The expression of Ki67 protein in each groups of cells was detected by immunocytochemistry.Results 1 According to the virus transfection instructions,ADAM17 overexpressed lentivirus(LV8N-ADAM17)was successfully transfected into MCF-7 cells,and the transfection efficiency was about 100%.2 The expression levels of ADAM17 mRNA in MCF-7 cells was detected by Real-time PCR,the expression of ADAM17 mRNA in blank control group,transfection control group group and transfection group was 1.03±0.14,1.18±0.10 and 1.81±0.06,respectively.The expression of ADAM17 mRNA in transfection group was significantly higher than that in blank control group and the transfection control group(P<0.05).However,there was no significant difference between the transfection control group and the blank control group(P>0.05).3 The expression levels of ADAM17 protein in MCF-7 cells was detected by Western blotting,the expression of ADAM17 protein in blank control group,transfection control group group and transfection group was 0.518±0.066,0.544±0.059 and 0.866±0.083,respectively.The expression of ADAM17 protein in transfection group was significantly higher than that in blank control group and the transfection control group,and the difference was statistically significant(P<0.05).However,there was no significant difference between the transfection control group and the blank control group(P>0.05).4 The result of MTT showed that the optical density of transfection group was 0.328±0.008,0.702±0.026 and 1.166±0.051 at 24 h,48h and 72 h,respectively.At 24 h,48h,72 h time points,there was a significant difference between the transfection group and the blank control group and the transfection control group(P<0.05).However,there was no significant difference between the transfection control group and the blank control group(P>0.05).5 The results of flow cytometry showed that compared with the blank control group and the transfection control group,the proportion of stationary phase in the transfection group decreased significantly,while the proportion of proliferative phase increased significantly.The statistical difference(P<0.05)showed that the proliferation of transfected cells was active.However,there was no significant difference between the transfection control group and the blank control group(P>0.05).6 The expression of Ki67 protein in each groups of cells was detected by immunocytochemistry.The staining score of Ki67 protein in control group,transfection control group group and transfection group was 6.01±0.77,6.72±0.53 and 8.29±0.48,respectively.The expression of Ki67 protein in transfection group was significantly higher than that in blank control group and the transfection control group(P<0.05).However,there was no significant difference between the transfection control group and the blank control group(P>0.05).Conclusions ADAM17 gene overexpression can enhance the ability of proliferation in MCF-7 breast cancer cells.