Effects of Midkine on cell proliferation ,invasion and epithelium mesenchymal transition of breast cancer cells

doi:10.3969/j.issn.1002-7386.2017.01.001

Abstract：Objective To investigate the effects of Midkine on cell proliferation , invasion and epithelium mesenchymal transition ( EMT ) in breast cancer .Mte hods The expression of midkine was silenced by lentivirus-mediated shRNAs interference technique ,and the high-expression gene of Midkine was used in normal breast epidermal cells .Western blotting was used to detect the expression levels of Midkine in normal breast epidermal cells ( BT549 ) and breast cancer cells ( HMLE) after shRNAs interference.The flow cytometry,Transwell chamber experiment and cell scratch assay were used to analyze the effects of Midkine gene on cell growth , proliferation,invasion and metastasis in normal breast epidermal cells and breast cancer cells .Results The expressions of Midkine protein were observed only in mesenchymal cells of breast cancer including BT549,MDA-MB157 and MDA-MB436, however, which were not observed in normal cells and epithelia cells of brease cancer .The two kinds of shRNA in BT 549 cells decreased the expression efficiency of Midkine protein by 100% and 90%,respectively .The crystal violet staining showed that the growth speed of BT 549 cells in Midkine-shRNAs transfection group was significantly decreased , as compared with that in negative control group ( P <0.01).The flow cytometry showed that the proliferation index of BT549 cells in Midkine-shRNAs transfection group was (36.06 ±2.12)%,(32.06 ±3.46)%, respectively,which was obviously lower than that in control group [(53.06 ±3.68)%].Meanwhile the expressions of EMT markers-B-catenins and N-cadherin were increased in BT549 cells in Midkine-shRNAs transfection group .Transwell chamber experiment showed that the cell count of BT 549 migration was 0 and 7 ±4/high power field in the two Midkine-shRNA transfection groups ,moreover, the invasion cell count was 38 ±7 and 46 ±8/high power field, which was significantly lower than the migration cell count (178 ± 14/high power field ) in control group ,and than the invasion cell count (232 ±35/high power field) .Moreover the phenotype of EMT was observed in normal breast epithelial cells that expressed Midkine protein highly , and the down-regulation of expressions of of EMT markers-E-cadherin and B-catenin was observed in HMLE cells that expressed Midkine protein highly .The cell scratch assay showed that the migration lenth in HMLE cells that expressed Midkine protein highly was significantly longer than that in control group .Conclusion To reduce the expression levels of Midkine in BT 549 cells can inhibit the migration and invasion of BT 549 cells, at the same time,which can up-regulate obviously the expression levels of epithelial mesenchymal protein markers-B-catenin and N-cadherin.Therefore,in tumorpatients with high expression of Midkine,tumor's invasion and metastasis can be inhibited by inhibiting the expression of Midkine protein.