PIN1反义核酸对乳腺癌MCF-7细胞增殖及周期的影响

Effect of PIN1 Antisense Nucleotide on Cell Proliferation and Cycle of Human Mammary Cancer Cell Line MCF-7

目的本研究利用PIN1反义核酸阻断乳腺癌MCF-7细胞中PIN1表达,观察其对增殖及周期的影响。方法构建PIN1反义核酸真核表达质粒pPIN1as,用脂质体转染法将重组质粒转染MCF-7细胞,G418筛选稳定表达重组质粒的克隆,RT-PCR检测PIN1基因表达水平,Western blot检测PIN1蛋白的表达,MTT检测细胞增殖状况,流式细胞术检测细胞周期。结果稳定表达PIN1反义核酸的MCF-7细胞内PIN1基因表达在mRNA水平和蛋白水平都显著降低;MTT实验显示MCF-7PINas细胞的增殖速度较MCF-7细胞明显减慢(P<0.05),但FCM显示MCF-7PINas细胞和MCF-7细胞的周期分布差异无统计学意义(P>0.05)。结论阻断PIN1可以显著抑制乳腺癌MCF-7细胞的增殖活性,提示PIN1可能成为乳腺癌基因治疗的新的靶点。

Objective To observe the effect of PIN1 antisense nucleotide on cell proliferation and cycle of human mammary cancer cell line MCF-7. Methods The eukaryotic vector named pPINas, expressing PIN1 antisense nucleotide, was constructed. MCF-7 was transfected with pPINas and selected in the culture with G418. The selected clone MCF-7PINas was checked for expression of PIN1 by RT-PCR and Western blot. The proliferation of ceils and the change of cell cycle in clone MCF-7PINas were investigated by MTT and Flow Cytometry respectively. Results The expression of PIN1 at mRNA and protein level in MCF-7PINas clone was down-regulated remarkably. MTT showed the proliferation of MCF- 7PINas was retarded obviously contrasting to MCF-7（P〈0. 05）. But the cell cycle distribution had no significant change（P〉0. 05）. Conclusion Since blocking PIN1 expression with antisense nucleotide could depress the proliferation activity of MCF-7 remarkably, PIN1 may be a potential target of gene therapy for human mammary cancer.