TRAIL通过降低EGFR的表达水平抑制乳腺癌MDA-MB-231细胞的侵袭

TRAIL suppresses the invasion capability of human breast cancer MDA-MB-231 cells through reducing EGFR expression

目的:探讨肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)抑制乳腺癌MDA-MB-231细胞侵袭能力的可能机制。方法:Western blotting、Real-time PCR分别检测rsTRAIL处理对MDA-MB-231细胞内EGFR、磷酸化转录因子IκBα(p-IκBα)和miR-146a表达的影响。向MDA-MB-231细胞转染miR-146a mimics、miR-146a inhibitor,Western blotting检测miR-146a对MDA-MB-231细胞中EGFR表达水平的影响。Transwell实验检测rsTRAIL、miR-146a和EGFR对MDA-MB-231细胞侵袭能力的影响。结果:20 ng/ml rsTRAIL显著抑制MDA-MB-231细胞中EGFR的表达(6 h,t=4.35,P<0.05;12 h,t=8.609,P<0.01;24 h,t=10.84,P<0.01),提高p-IκBα(6 h,t=-4.201,P<0.05;12h,t=-15.805,P<0.01;24 h,t=-35.921,P<0.01)和miR-146a的表达水平(6 h,t=-4.67,P<0.05;12 h,t=-11.635,P<0.01;24 h,t=-15.8,P<0.01),并且呈时间依赖性。在MDA-MB-231细胞中转染miR-146a mimics显著抑制EGFR的表达(t=6.25,P<0.01);反之,转染miR-146a inhibitor则促进细胞内EGFR的表达(t=-3.674,P<0.05)。rsTRAIL处理(t=7.108,P<0.01)、转染miR-146a mimics或si EGFR(t=6.051,P<0.01;t=5.245,P<0.01)均导致细胞的侵袭能力显著下降。结论:rsTRAIL通过特异性增加miR-146a的表达水平靶向降低EGFR的表达从而抑制乳腺癌MDA-MB-231细胞的侵袭能力。

Objective: To investigate the potential mechanism by which tumor necrosis factor-related apoptosis-inducing ligand( TRAIL) inhibits the invasion capability of breast cancer MDA-MB-231 cells. Methods: After treating MDA-MB-231 cells with rsTRAIL,the expression of EGFR and p-IκBα were examined by immunoblotting,and the level of miR-146 was measured by real-time quantitative PCR. Immunoblotting was also used to detect the effect of miR-146 a on EGFR expression. Transwell assay was carried out to assess the effects of rsTRAIL,miR-146 a and EGFR on the invasion ability of MDA-MB-231 cells. Results: In MDA-MB-231 cells treated for 6,12,and 24 hours,rsTRAIL( 20 ng / ml) significantly suppressed the expression of EGFR( 6 h,t = 4. 35,P < 0. 05; 12 h,t = 8. 609,P < 0. 01; 24 h,t = 10. 84,P < 0. 01),increased the level of the phosphorylated IκBα( 6 h,t =- 4. 201,P < 0. 05; 12 h,t =- 15. 805,P < 0. 01; 24 h,t =- 35. 921,P < 0. 01), and upregulated the expression of miR-146a( 6 h, t =- 4. 67,P < 0. 05; 12 h,t =- 11. 635,P < 0. 01; 24 h,t =- 15. 8,P < 0. 01),and on time dependent. Compared with that in control cells,the level of EGFR( t = 6. 25,P < 0. 01) was significantly decreased in MDA-MB-231 cells transfected with miR-146 a mimics,whereas in the same cells transfected with miR-146 inhibitor the expression of EGFR promoted( t =- 3. 674,P <0. 05). Furthermore,transfection with rsTRAIL,as well as transfection with miR-146 a mimics or si EGFRall dramatically decreased the invasion ability of MDA-MB-231 cells( t = 7. 108,P < 0. 01; t = 6. 051,P < 0. 01; t = 5. 245,P < 0. 01respectively). Conclusion: rsTRAIL specifically suppresses EGFR-dependent invasion capability of human breast cancer through inducing increased expression of miR-146 a.