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目的探讨短发夹RNA(shRNA)靶向沉默DLL4基因对MCF-7细胞凋亡的诱导及对化疗药物多西他赛的增敏作用。方法针对DLL4基因的序列设计具有特异性的shRNA,经脂质体转染MCF-7细胞,作为实验组,空脂质体转染的MCF-7细胞为脂质体组,未做任何处理的MCF-7细胞为对照组。采用免疫细胞化学方法检测3组细胞DLL4蛋白的表达情况;采用流式细胞仪检测3组细胞凋亡率及细胞周期改变;采用噻唑蓝(methyl-thiazoyl-tetrazolium bromide,MTT)法测定3组细胞的增殖情况及对多西他赛的敏感性。结果实验组平均光密度值及阳性面积率均低于对照组和脂质体组(P<0.01);实验组细胞在24 h、48 h及72 h时间点A值均低于对照组和脂质体组(P<0.01);转染后24 h、48 h、72 h及96 h,实验组细胞凋亡率高于对照组和脂质体组(P<0.05);RNA干扰(RNAi)沉默DLL4基因后,实验组G2/M期细胞比例高于对照组和脂质体组(P<0.05);实验组的IC50值较对照组和脂质体组低(P<0.05)。结论 RNAi技术抑制DLL4基因的表达能够抑制MCF-7细胞的增殖、诱导细胞凋亡及增强细胞对多西他赛作用的敏感性。DLL4可能会成为乳腺癌治疗的重要靶点。
Objective To investigate the induction of apoptosis by short hairpin RNA (shRNA) targeting DLL4 gene in MCF-7 cells and its sensitization to docetaxel. Methods Specific shRNAs were designed according to the sequence of DLL4 gene and transfected into MCF-7 cells by liposome. As experimental group, empty liposome-transfected MCF-7 cells were liposome group without any treatment MCF-7 cells as control group. The expression of DLL4 protein in three groups of cells was detected by immunocytochemistry. The apoptosis rate and cell cycle of three groups were detected by flow cytometry. Three groups of cells were determined by methyl thiazolyl tetrazolium bromide (MTT) Proliferation and Sensitivity to Docetaxel. Results The average optical density and the positive area ratio of the experimental group were lower than those of the control group and the liposome group (P <0.01). The A value of the experimental group at 24 h, 48 h and 72 h was lower than that of the control group and lipid (P <0.01). The apoptotic rate of the experimental group was higher than that of the control group and the liposome group at 24 h, 48 h, 72 h and 96 h after transfection (P <0.05) After silenced DLL4 gene, the proportion of cells in G2 / M phase in experiment group was higher than that in control group and liposome group (P <0.05). The IC50 value in experimental group was lower than that in control group and liposome group (P <0.05). Conclusion RNAi can inhibit the proliferation of MCF-7 cells, induce apoptosis and enhance the sensitivity of cells to docetaxel by inhibiting the expression of DLL4 gene. DLL4 may be an important target for the treatment of breast cancer.