果糖-1,6-二磷酸酶1过表达抑制他莫昔芬耐药的乳腺癌细胞迁移并促进细胞凋亡

Over-expression of fructose-1,6-bisphosphatase 1 gene inhibits the migration of breast cancer tamoxifen-resistant cells and induces apoptosis

目的 :探讨过表达果糖-1,6-二磷酸酶1(fructose-1,6-bisphosphatase 1,FBP 1)基因对他莫昔芬耐药性人乳腺癌MCF-7R细胞迁移和凋亡的影响。方法 :首先采用实时荧光定量PCR和蛋白质印迹法检测人乳腺癌亲本敏感细胞MCF-7和他莫昔芬耐药细胞MCF-7R中FBP1 mRNA和蛋白的表达水平。然后,采用脂质体转染法将FBP1过表达重组质粒转染至耐药细胞MCF-7R中,再应用实时荧光定量PCR和蛋白质印迹法检测FBP1 mRNA和蛋白的表达水平,以验证FBP1基因过表达效果。接着,倒置光学显微镜下观察FBP1过表达后MCF-7R细胞的形态学变化;FCM法和细胞划痕愈合实验分别检测细胞凋亡及迁移能力的变化;蛋白质印迹法检测凋亡相关蛋白Bcl-2和Bax以及上皮-间质转化标志物E-cadherin和vimentin的表达变化。结果 :对他莫昔芬耐药的人乳腺癌细胞MCF-7R中FBP1 mRNA和蛋白的表达水平均比其亲本细胞MCF-7明显降低(P值均<0.01)。FBP1过表达重组质粒转染后,MCF-7R细胞中FBP1 mRNA及蛋白的表达水平均明显上调(P值均<0.01),MCF-7R细胞形态由间质细胞样向上皮细胞样转变,细胞凋亡率明显升高(P<0.01),迁移能力则明显降低(P<0.01)。FBP1过表达的MCF-7R细胞中Bcl-2和vimentin蛋白的表达水平明显下降(P值均<0.01),Bax和E-cadherin蛋白的表达水平则明显升高(P值均<0.01)。结论 :FBP1基因过表达可明显抑制人乳腺癌他莫昔芬耐药细胞MCF-7R的迁移和上皮-间质转化,并通过下调Bcl-2表达和上调Bax表达来促进肿瘤耐药细胞的凋亡。

Objective： To investigate the effects of over-expressing fructose-1,6-bisphosphatase 1（FBP 1） gene on migration and apoptosis of tamoxifen-resistant MCF-7 human breast cancer cells.Methods： The levels of FBP1 mRNA and protein in human breast cancer tamoxifen-sensitive MCF-7 cells and tamoxifen-resistant MCF-7R cells were measured by real-time fluorescent quantitative PCR and Western blotting, respectively. Then, the FBP1 over-expression plasmids were transfected into MCF-7R cells by lipofectin, and the expression levels of FBP1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting in order to verify the over-expression of FBP 1 gene. After that, the morphology of MCF-7R cells with FBP1 over-expression was observed under an inverted optical microscope, the abilities of cell migration and apoptosis were measured by cell wound healing experiment and FCM assay, respectively. Furthermore, the expressions of apoptosis-associated proteins Bcl-2 and Bax as well as the epithelial-mesenchymal transition（EMT） markers E-cadherin and vimentin were detected by Western blotting.Results： The mRNA and protein levels of FBP1 in human breast cancer tamoxifen-resistant MCF-7R cells were lower than those in tamoxifen-sensitive MCF-7 cells（both P〈0.01）. After the transfection of FBP1 over-expression plasmids, the mRNA and protein expressions of FBP1 were significantly up-regulated in MCF-7R cells（both P〈0.01）, MCF-7R cells had a change from mesenchymal-like morphology to epithelial-like morphology, the apoptotic index of MCF-7R cells was increased（P〈0.01）, but the ability of cell migration was decreased（P〈0.01）. Moreover, the protein expressions of Bcl-2 and vimentin were obviously down-regulated（both P〈0.01）, while the protein expressions of Bax and E-cadherin were significantly up-regulated（both P〈0.01）.Conclusion： The over-expression of FBP 1 gene can significantly inhibit cell migration and EMT in human breast cancer tamoxifen-resistant MCF-7R cells, and promote apoptosis through decreasing Bcl-2 expression and increasing Bax expression.