Zhou Ji-hong, Tang Xiao-yan, Zhao Rui, Wang Hui, Xia Jun
Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Mar;28(3):260-4.
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.
(1) A recombinate pLNCX-RI and an empty pLNCX were transferred into MDA-MB-231 cells by using Lipofectamin(TM) 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.
(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).
(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.
将RI基因转染至RI基因相对低表达的人乳腺癌细胞系MDA-MB-231中，研究RI基因对MDA-MB-231细胞凋亡及侵袭能力的影响。
（1）采用脂质体2000将重组质粒pLNCX-RI及空质粒pLNCX转入MDA-MB-231细胞。转染后用G418筛选阳性克隆并扩大培养，采用RT-PCR及Western blot法分析转染前后MDA-MB-231细胞中RI mRNA及蛋白的表达情况。（2）采用Transwell实验、流式细胞术（FCM）检测RI表达对转染细胞凋亡及侵袭能力的影响。（3）采用RT-PCR及Western blot检测MDA-MB-231细胞中survivin的mRNA及蛋白表达，采用Western blot检测MDA-MB-231细胞中Caspase-3的蛋白表达，初步研究RI诱导乳腺癌细胞凋亡的相关机制。（4）采用RT-PCR及Western blot检测MDA-MB-231细胞中CD24的mRNA及蛋白表达，初步研究RI抑制乳腺癌细胞侵袭的机制。
（1）利用脂质体2000成功将RI基因转染至MDA-MB-231细胞，筛选出高表达RI的亚克隆细胞系MDA-MB-231/pLNCX-RI。（2）与MDA-MB-231及MDA-MB-231/pLNCX细胞相比，MDA-MB-231/pLNCX-RI细胞的FCM及Transwell实验结果显示：细胞凋亡率明显增加（P<0.01），穿膜细胞数减少（P<0.01）。MDA-MB-231/pLNCX-RI细胞中Survivin的mRNA及蛋白表达降低，Caspase-3被激活（P<0.01）。MDA-MB-231/pLNCX-RI细胞中CD24的mRNA及蛋白表达降低（P<0.01）。
（1）外源性RI表达可能通过抑制Survivin表达、激活Caspase-3促进人乳腺癌细胞MDA-MB-231凋亡。（2）外源性RI表达可能通过抑制CD24表达抑制MDA-MB-231细胞的侵袭能力。
