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.

辽宁省自然科学基金资助项目（No.2013022051）