肝素结合细胞因子影响乳腺癌细胞的增殖、侵袭和上皮细胞间质化的功能研究

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

目的探讨肝素结合细胞因子(Midkine)的表达对乳腺癌细胞的增殖和侵袭的作用。方法采用慢病毒介导的shRNA干扰技术在乳腺癌细胞中沉默Midkine的表达,及利用慢病毒高表达Midkine基因在正常乳腺表皮细胞中。利用蛋白质印迹(Western blot)法检测shRNA干扰或高表达后的正常乳腺表皮细胞和乳腺癌细胞中Midkine蛋白的表达情况。利用流式细胞仪技术、Transwell小室实验、及细胞划痕实验,分析Midkine基因高低表达对正常乳腺表皮细胞和乳腺癌细胞生长、增殖、侵袭及转移的影响。结果首先观察了不同乳腺癌细胞中Midkine的表达情况。其中Midkine只有在BT549、MDA-MB157和MDA-MB436间充质乳腺癌细胞(Mesenchymal cell)中表达,而正常细胞和上皮细胞乳腺癌细胞(Epithelial cell)中不表达。BT549细胞中两种不同shRNA降低Mdikine蛋白表达的效率分别达到100%和90%。HMLE正常乳腺上皮细胞中Midkine高表达效率非常高。结晶紫染色法检测细胞增殖实验结果显示,Midkine-shRNAs转染组BT549细胞的生长速度较阴性对照组降低(P<0.01)。流式细胞仪检测结果显示,Midkine-shRNAs转染组BT549细胞的增殖指数为(36.06±2.12)%和(32.06±3.46)%,明显低于对照组53.06±3.68%。同时观察到EMT标识物B连环蛋白(β-catenin)、N钙粘蛋白(N-cadherin)在Midkine-shRNAs转染组BT549细胞中表达上调。Transwell小室实验显示两个Midkine-shRNA转染组BT549细胞迁移(Migration)细胞数为零和(7±4.4)个/高倍视野,它们的侵袭(Invasion)细胞数为(38±7)和(46±8)个/高倍视野,明显低于对照组的迁移(Migration)细胞数(178±14)个/高倍视野,侵袭(Invasion)细胞数(232±35)个/高倍视野。同时也观察到高表达Midkine正常乳腺上皮细胞表现出间质化(EMT)的表型,EMT标识物E钙粘蛋白(E-cadherin)、连环蛋白(β-catenin)在Midkine高表达HMLE细胞中表达下调的现象。细胞划痕实验显示Midkine高表达HMLE细胞组的迁移距离明显高于对照组。结论在BT549细胞中降低Midkine的表达,可抑制BT549细胞的迁移和侵袭,同时可明显上调对上皮细胞间质化标志蛋白β-catenin和N-cadherin表达,提示在Midkine高表达的肿瘤患者中,通过抑制其蛋白表达可能抑制肿瘤的转移和侵袭。

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＆#39;s invasion and metastasis can be inhibited by inhibiting the expression of Midkine protein.