干扰MAD2L1基因表达对乳腺癌细胞凋亡的影响及机制

目的 探讨干扰有丝分裂阻滞缺陷2样蛋白1(MAD2L1)基因表达对乳腺癌(BC)细胞凋亡的影响及其机制。方法 采用qRT-PCR检测正常乳腺上皮细胞系MCF-10A和4种BC细胞系(MDA-MB-231、MCF-7、SK-BR-3和BT-20)中MAD2L1 mRNA表达水平。将MAD2L1 siRNA转染至MDA-MB-231细胞中，qRT-PCR和Western blotting检测细胞中MAD2L1 mRNA和蛋白表达水平；MTT检测细胞增殖能力；流式细胞术法检测细胞凋亡率；Western blotting检测细胞中Bax、Bcl-2、cleaved-caspase-3、p-p38MAPK(Thr180/Thr182)和p38MAPK等蛋白表达水平。采用p38MAPK抑制剂SB203580联合处理上述细胞，Annexin V-FITC/PI法检测细胞凋亡率的变化；Western blotting检测Bax、Bcl-2、cleaved-caspase-3、p-p38MAPK和p38MAPK表达水平的变化。结果 BC细胞系中MAD2L1 mRNA表达水平高于MCF-10A细胞，其中M...

Effects and mechanism of interfering MAD2L1 gene expression on the apoptosis of breast cancer cells

Objective To explore the effects of interference with mitotic arrest deficient 2-like protein 1(MAD2L1)gene expression on breast cancer(BC)cell apoptosis and the mechanism. Methods The mRNA expressions of MAD2L1 in normal breast epithelial cell line MCF-10A and BC cell lines, including MDA-MB-231, MCF-7, SK-BR-3 and BT-20, were detected with qRT-PCR. After MAD2L1 siRNA was transfected into MDA-MB-231 cells, the mRNA and protein expressions of MAD2L1 were detected with qRT-PCR and Western blotting; cell proliferation ability was measured with MTT; cell apoptosis was detected with Annexin V-FITC/PI; expressions of Bax, Bcl-2, cleaved-caspase-3, p-p38MAPK(Thr180/Thr182)and p38MAPK were determined with Western blotting. After the above cells were treated with the p38MAPK inhibitor SB203580 in combination, the changes in apoptosis rate were detected with flow cytometry, and changes in the expressions of Bax, Bcl-2, cleaved-caspase-3, p-p38MAPK and p38MAPK were detected with Western blotting. Results The mRNA expression of MAD2L1 in BC cell line, especially in MDA-MB-231 cells, was significantly higher than that in MCF-10A cells. Interference with MAD2L1 gene reduced the mRNA and protein expressions of MAD2L1 in MDA-MB-231 cells(t_mRNA=10.51, P_mRNA<0.001; t_protein=18.30, P_protein<0.001), inhibited cell proliferation(F_group=243.36, F_time=44.00, F_{group × time}=9.881, all P<0.001), promoted cell apoptosis(t=9.10, P<0.001), up-regulated the protein expressions of Bax, cleaved-caspase-3 and p-p38MAPK(t_Bax=15.05, P_Bax<0.001; t_{cleaved-caspase-3}=5.26, P_{cleaved-caspase-3}=0.006; t_p-p38MAPK=28.46, P_p-p38MAPK<0.001), and down-regulated the protein expression of Bcl-2(t_Bcl-2=14.23, P_Bcl-2<0.001). However, SB203580 treatment inhibited the induction of apoptosis by MAD2L1 gene interference on MDA-MB-231 cells(P=0.002). Conclusion Interfering with the expression of MAD2L1 gene can inhibit the proliferation of MDA-MB-231 cells and induce apoptosis, and the mechanism may be related to the activation of the p38MAPK signaling pathway.