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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。
AIM: To investigate the effects of fructose-1,6-bisphosphatase 1 (FBP1) overexpression on migration and apoptosis of tamoxifen-resistant human breast cancer MCF-7R cells . Methods: The expression of FBP1 mRNA and protein in MCF-7 cells and MCF-7R cells were detected by real-time fluorescence quantitative PCR and Western blotting. Then, FBP1 overexpression recombinant plasmid was transfected into MCF-7R cells by lipofection method. The expression of FBP1 mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blotting to verify the effect of FBP1 gene over-expression Express the effect. The morphological changes of MCF-7R cells after FBP1 overexpression were observed under an inverted light microscope. The changes of apoptosis and migration ability were detected by FCM and cell scratch healing assay, respectively. Western blotting was used to detect the expression of apoptosis related protein Bcl-2 And Bax, as well as the expression of epithelial-mesenchymal transition markers E-cadherin and vimentin. Results: The expression levels of FBP1 mRNA and protein in MCF-7R cells resistant to tamoxifen were significantly lower than those in MCF-7 cells (all P <0.01). After transfection with FBP1 overexpression plasmid, the expression levels of FBP1 mRNA and protein in MCF-7R cells were significantly increased (all P <0.01). The morphology of MCF-7R cells was changed from stromal cells to epithelial cells, Mortality increased significantly (P <0.01), migration ability was significantly lower (P <0.01). The expression of Bcl-2 and vimentin in FBP1-overexpressing MCF-7R cells was significantly decreased (P <0.01), while the expression of Bax and E-cadherin was significantly increased (all P <0.01). CONCLUSION: FBP1 gene overexpression can significantly inhibit the migration and epithelial-mesenchymal transition of human breast cancer tamoxifen-resistant cells MCF-7R and promote the apoptosis of drug-resistant cells by down-regulating Bcl-2 expression and up-regulating Bax expression Death.