5-脂氧合酶激活蛋白小干扰RNA诱导人乳腺癌细胞凋亡的初步研究

5-Lipoxygenase Activating Protein Knockdown Using siRNA Induces Apoptosis in Human Breast Cancer Cells

目的:研究5-脂氧合酶激活蛋白(FLAP)的表达抑制对乳腺癌细胞凋亡的诱导作用。方法:通过小干扰RNA(siRNA)抑制乳腺癌细胞MDA-MB-231中FLAP的表达,用流式细胞仪检测膜联蛋白(annexin)-Ⅴ标记的早期凋亡细胞,用Western印迹检测细胞凋亡相关蛋白的水平。结果:转染了FLAP siRNA的乳腺癌细胞,24 h后FLAP的表达被抑制,17%的细胞出现早期凋亡;48 h时早期凋亡细胞增加到32.1%;72 h时早期凋亡细胞下降到13.8%,而死亡或凋亡晚期细胞占到61.3%。在细胞凋亡过程中,Bcl-2水平下降,而细胞色素c、胱冬蛋白酶(caspase)-3的水平逐渐增高。结论:FLAP的表达抑制可以诱导乳腺癌细胞通过Bcl-2和胱冬蛋白酶-3途径发生凋亡。

Objective： To investigate the roles of 5-1ipoxygenase activating protein（FLAP） in apoptosis and cell proliferation by FLAP knockdown in human breast cells. Methods： FLAP was knockdowned by siRNA for 24, 48 and 72 hours in human breast cancer cells MDA-MB-231. Cell viability was investigated with cell counting using hemocytometery and trypan blue staining. Apoptosis was examined with Annexin-V and flow cytometery. The levels of Bcl-2, caspase-3 and some other related proteins were detected by Western blotting. Results： FLAP was decreased apparently at 24 hours, dramatically at 48 and 72 hours by siRNA. Along with the treatment of FLAP siRNA, cell viability was decreased gradually. Flow cytometery showed that 17% and 32.1% cells were in early apoptosis after 24 and 48 hours, respectively, and 61.3% cells went to late apoptosis or death after 72 hours. Apoptosis in MDA-MB-231 cells was accompanied by release of cytochrome c, activation of caspase-3, and specific proteolytic cleavage of poly-（ADP-ribose） polymerase（PARP）. Conclusion： The data suggests that FLAP knockdown exerts growth inhibition on highly invasive estrogen-nonresponsive human breast cancer cells through apoptosis induction, and that FLAP may be chosen as a new target molecular for the anti-cancer drug products.