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目的:将核糖核酸酶抑制因子(RI)转染乳腺癌细胞株MDA-MB-231中,探讨RI对细胞凋亡和侵袭的影响。方法:(1)应用脂质体LipofectamineTM2000分别将重组表达载体pLNCX-RI和空载体pLNCX导入MDA-MB-231细胞,G418筛选阳性克隆并扩增传代。RT-PCR和Western blot检测RI表达。(2)流式细胞术、Transwell细胞侵袭实验分别观察RI对乳腺癌细胞凋亡、侵袭的影响。(3)RT-PCR和West-ern blot法检测Survivin mRNA和蛋白的表达,Western blot方法检测Caspase-3蛋白的活化情况,初步探讨RI诱导乳腺癌细胞凋亡的相关机制。(4)RT-PCR和Western blot法检测CD24mRNA和蛋白的表达,初步探讨RI抑制细胞侵袭的机制。结果:(1)成功构建表达外源性RI的亚克隆细胞系MDA-MB-231/pLNCX-RI。(2)与未转染的MDA-MB-231细胞及转染空载体的MDA-MB-231/pLNCX细胞相比,MDA-MB-231/pLNCX-RI细胞凋亡率增加(P<0.01);细胞穿膜数减少(P<0.01);Survivin mRNA和蛋白的表达量下降(P<0.01),且能活化Caspase-3;CD24 mRNA和蛋白的表达量亦下降(P<0.01)。结论:(1)外源性RI基因在乳腺癌MDA-MB-231细胞中的稳定表达可以通过抑制Survivin的表达和激活Caspase-3促进细胞凋亡,并能通过抑制CD24表达降低细胞侵袭力。
Objective: To investigate the effect of RI on apoptosis and invasion in breast cancer cell line MDA-MB-231 by transfecting ribonuclease inhibitor (RI). Methods: (1) The recombinant plasmid pLNCX-RI and empty vector pLNCX were transfected into MDA-MB-231 cells respectively by LipofectamineTM2000. The positive clones were selected by G418 and passaged for amplification. RI expression was detected by RT-PCR and Western blot. (2) Flow cytometry and Transwell invasion assay were used to observe the effect of RI on apoptosis and invasion of breast cancer cells. (3) The expression of Survivin mRNA and protein were detected by RT-PCR and West-ern blot. The activation of Caspase-3 protein was detected by Western blot and the mechanism of RI-induced apoptosis in breast cancer cells. (4) The expression of CD24mRNA and protein were detected by RT-PCR and Western blot, and the mechanism of RI inhibition on cell invasion was discussed. Results: (1) The subclone cell line MDA-MB-231 / pLNCX-RI expressing exogenous RI was successfully constructed. (2) The apoptosis rate of MDA-MB-231 / pLNCX-RI cells was increased (P <0.01) compared with untransfected MDA-MB-231 cells and MDA-MB-231 / pLNCX cells transfected with empty vector (P <0.01). The expression of Survivin mRNA and protein was decreased (P <0.01), and Caspase-3 was also activated. The expression of CD24 mRNA and protein also decreased (P <0.01). Conclusion: (1) Stable expression of exogenous RI gene in breast cancer MDA-MB-231 cells can promote apoptosis by inhibiting the expression of Survivin and activating Caspase-3, and can reduce the invasiveness of cells by inhibiting the expression of CD24.