c-FLIP-L影响乳腺癌MDA-MB-231细胞对TRAIL致凋亡作用的敏感性

Effect of c-FLIP-L on the sensitivity of breast cancer MDA-MB-231 cells to TRAIL-induced apoptosis

目的:观察c-FLIP-L对TRAIL诱导乳腺癌MDA-MB-231细胞凋亡的影响及其具体机制。方法:构建靶向c-FLIP-L的siRNA质粒,转染乳腺癌MDA-MB-231细胞,RT-PCR、Weston blotting验证基因抑制效果。实验分空白对照组、TRAIL组、c-FLIP-L siRNA组和c-FLIP-L siRNA+TRAIL组共4组,MTT法检测各组MDA-MB-231细胞的增殖情况,流式细胞术检测MDA-MB-231细胞凋亡率,Transwell实验检测MDA-MB-231细胞侵袭能力,RT-PCR、Western blotting检测MDA-MB-231细胞中c-FLIP-L、caspase-3、caspase-8、MMP-2、MMP-9的表达。结果:靶向c-FLIP-L的siRNA质粒转染至乳腺癌细胞株可有效抑制c-FLIP-L mRNA[(37.12±3.02)vs(183.21±8.31),(174.65±10.06);P<0.05]及其蛋白的水平。c-FLIP-L siRNA和TRAIL联合处理MDA-MB-231细胞,与两者单独处理相比,细胞增殖抑制率显著升高[72 h时,(75.51±2.01)%vs(33.75±1.60)%,(34.31±2.01)%;均P<0.05],凋亡率显著升高[72 h时,(76.30±4.11)%vs(38.95±2.14)%,(29.28±1.66)%;均P<0.05],对细胞侵袭能力的抑制也显著增强。c-FLIP-L siRNA和TRAIL联合处理后,与两者单独处理相比,乳腺癌细胞中casepase-3、casepase-8的表达显著增强,而MMP-2、MMP-9的表达明显减弱。结论:抑制c-FLIP-L表达能够增强MDA-MB-231细胞对TRAIL的敏感性,从而提高诱导肿瘤细胞凋亡的能力,其机制可能与caspase-3、caspase-8的表达上调和MMP-2、MMP-9的表达下调有关。

Objective ： To study the effect of c-FLIP-L on TRAIL-induced breast cancer apoptosis. Methods： c-FLIP-L was silenced in breast cancer MDA-MB-231 cells by siRNA. After c-FLIP-L silencing, cell proliferation was assessed by MTT assay, cell apoptosis by Annexin-V FITC/PI double staining flow cytometry, invasive potential by matrigel invasion assay, and protein and mRNA levels of c-FLIP-L, caspase-3, caspase-8, MMP-2, MMP-9 in transfected cells by Western blotting and RT-PCR respectively. Results： The sequence-specific siRNA significantly decreased c-FLIP-L mRNA （3712±3.02 vs 183.21±8.31, 174.65±10.06; P 〈0.05） and protein levels as compared with the control. At 72 h after treatment, c-FLIP-L siRNA and TRAIL, either in combination or each alone, significantly inhibited proliferation （[75.51±2.01]% vs [33.75±1.60]%, [34.31±2.01]%; P 〈0.01）, induced apoptosis （[76.30±4.11]% vs [38.95±2.14]%, [29.28±1.66]%; P 〈0.05）, decreased the invasive capacity. Enhanced casepase-3 and casepase-8 expression,and inhibited MMP-2 and MMP-9 expression in MDA-MB-231 cells. Conclusion： Inhibition of c-FLIP-L expression may increase the sensitivity of breast cancer cells to TRAIL-induced apoptosis, possibly through enhancing the expression of caspase-3, caspase-8 and inhibiting the expression of MMP-2 and MMP-9.