核糖核酸酶抑制因子对人乳腺癌MDA-MB-231细胞凋亡和侵袭的影响

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。

Effects of ribonuclease inhibitor on apoptosis and invasion of human breast cancer MDA-MB-231 cells

AIM: RI gene is transfected into human breast cell line MDA-MB-231 which is relatively hypo-expression RI gene.To investigate how RI gene affect the cell apoptosis and invasion of MDA-MB-231.METHODS:(1) A recombinate pLNCX-RI and an empty pLNCX were transfered into MDA-MB-231 cells by useing LipofectaminTM 2000.After transfection,positive clones were screened with G418 and expanded by culture.RT-PCR and Western blot methods were used to analyze expression of RI mRNA and protein in MDA-MB-231 cells before and after transfection.(2) Transwell test,FCM test were used to search for the effects of RI expression on transfected cells apoptosis and invasion.(3)To study preliminarily the related mechanism by which RI induced breast cancer cell apoptosis,the mRNA and protein expression of survivin in MDA-MB-231 cells were examined by RT-PCR and Western blot,and protein expression of Caspase-3 in MDA-MB-231cells were examined by Western blot.(4) To investigate preliminarily the mechanism by which RI inhibited breast cancer cell invasion,the mRNA and protein expression of CD24 in MDA-MB-231 cells were examined by RT-PCR and Western blot.RESULTS:(1) The RI gene was transfected successfully to MDA-MB-231 by using LipofectamineTM2000,the subclone cell lines MDA-MB-231/pLNCX-RI which highly expressed RI were successfully selected.(2) Compared with MDA-MB-231 and MDA-MB-231/pLNCX cells,the results of FCM and transwell in MDA-MB-231/pLNCX-RI cells indicated: the percentage of cell apoptosis were obviously increased(P<0.01),penetrating membrane cells were decreased(P<0.01).The mRNA and protein expression of Survivin were degraded,and Caspase-3 was activated in MDA-MB-231/pLNCX-RI cells(P<0.01).The mRNA and protein expression of CD24 were degraded in MDA-MB-231/pLNCX-RI cells(P<0.01).CONCLUSION:(1) Exogenous RI expression may promote apoptosis in human breast cells MDA-MB-231 by inhibiting the expression of Survivin and activating caspase-3.(2) Exogenous RI expression may inhibit invasion in MDA-MB-231 by inhibiting expression of CD24.