小分子RNA干扰MTA1基因对乳腺癌MDA-MB-231细胞生物活性及相关蛋白表达的影响

Effect of interference of MTA1 by shRNA on the biological behaviors and expression of related proteins in breast cancer MDA-MB-231 cells

目的:采用短发夹状RNA(short hairpin RNA,shRNA)干扰技术沉默乳腺癌细胞MDA-MB-231中转移相关基因(metastasis-associated gene 1,MTA1)的表达,并观察其对15-脂氧合酶-2(15-lipoxygenase-2,15-LOX-2)、p53及bcl-2表达的影响。方法:将shRNA-MTA1载体质粒稳定转染MDA-MB-231细胞,MTT法检测细胞增殖抑制情况,FCM法检测细胞周期及凋亡情况,RT-PCR和Western印迹法检测MTAl、15-LOX-2、p53及bcl-2 mRNA和蛋白表达情况。结果:shRNA-MTA1能明显降低MTA1基因在MDA-MB-231细胞中的表达量;抑制MDA-MB-231细胞的增殖,诱导其凋亡,并使细胞被阻滞在G1期,与对照组相比,差异有统计学意义(P<0.01)。转染shRNA-MTA1可使MDA-MB-231细胞中15-LOX-2和p53 mRNA及蛋白的表达明显增强(P<0.01),而MTA1和bcl-2 mRNA表达明显减弱(P<0.01)。结论:MTA1基因可抑制乳腺癌MDA-MB-231细胞的增殖,诱导其凋亡,该作用可能与上调细胞中15-LOX-2和p53的表达,下调bcl-2的表达有关。

Objective:To silence the expression of metastasis-associated gene 1(MTA1) in breast cancer cell line MDA-MB-231 by using short hairpin small interfering RNA(shRNA) and observe its effects on expression of 15-lipoxygenase 2(15-LOX-2),p53 and bcl-2 proteins.Methods:The shRNA-MTA1 plasmid was stably transfected into MDA-MB-231 cells and the cell proliferation was evaluated using MTT assay.The cell cycle distribution and apoptosis were analyzed using flow cytometry.The mRNA and protein expression levels of MTAl,15-LOX-2,p53 and bcl-2 were determined using RT-PCR and Western blotting,respectively.Results:shRNA-MTAl significantly suppressed the expression of MTA1 gene in MDA-MB-231 cells,inhibited the proliferation,induced apoptosis,and arrested the cells in G1 phase.The difference was significant compared with control group(P0.01).Expression levels of 15-LOX-2 and p53 were significantly up-regulated but MTAl and bcl-2 were significantly down-regulated in MDA-MB-231 cells in shRNA-MTA1 group compared with the blank control group and negative control group(P0.01).Conclusion:Silencing MTA1 gene inhibited the proliferation and induced the apoptosis of MDA-MB-231 cells.This effect may be related with up-regulation of the expression of 15-LOX-2 and p53 and down-regulation of bcl-2.