siRNA沉默LOX基因对鼻咽癌细胞侵袭及迁移能力的影响

摘要

目的:探讨RNAi干扰赖氨酰氧化酶基因(LOX)后对鼻咽癌细胞5－8F侵袭及迁移能力的影响.方法:设计并构建针对LOX基因的RNAi片段,脂质体介导siRNA－LOX－01(siRNA－LOX－01组)和siRNA－LOX－02(siRNA－LOX－02组)转染5 －8F细胞,同时设非特异性干扰片段为对照组.应用实时荧光定量PCR、免疫印迹试验检测转染后各组LOX的mRNA和蛋白表达及上皮间质转化(EMT)相关指标E－cadher-in、N－cadherin、Vimentin、Snail、Slug、MMP9等的表达变化;细胞黏附实验、基底膜侵袭实验和细胞划痕实验分别研究LOX基因沉默后对5－8F细胞侵袭及迁移能力的影响.结果:与对照组相比,siRNA－LOX－01组和siRNA－LOX－02组LOX表达在mRNA及蛋白水平下降,细胞的迁移速率变慢,穿膜细胞数减少,细胞黏附率增加(均P＜0.05),E－cadherin的表达增加,N－cadherin、Vimentin、Snail、Slug及MMP9的表达减少.结论:干扰LOX能明显抑制鼻咽癌细胞的侵袭及迁移能力,作用机制可能与N－cadherin、Vimentin、Snail、Slug及MMP9蛋白表达下调有关,LOX有望成为治疗鼻咽癌的有效靶点.%Objective:To study the effects of LOX down－regulation on the migration and invasion of nasopharyn-geal carcinoma cell line 5－8F in vitro.Methods:siRNA(siRNA－LOX－01 and siRNA－LOX－02)that can tran-siently silenced LOX was designed,synthesized and transfected into 5－8F cells by lipofectamine 2000.Non－specific siRNA was transfected to be used as negative control.The efficiency of LOX depletion was determined by Real－time quantitative PCR and Western blot.Wound－healing assay,Matrigel invasion assay and the solid－phase adhesion as-say were performed to examine cell metastasis.The expressions of LOX,E－cadherin,N－cadherin,Vimentin,Snail,Slug and MMP9 were determined by Western blot.Results:Compared with cells in negative control group,transiently knocking down of LOX decreased transcription and expression in 5－8F cells showing by Real－time PCR and West-ern blot.The invasion and migration of the cells transfected with siRNA－LOX were much weaker than the control group(P＜0.05).The expressions of LOX,N－cadherin,Vimentin,Snail,Slug and MMP9 proteins in cells of LOX silence group were much lower while E－cadherin expression level was much higher than those in negative control.Conclusion:Down－regulation of LOX can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cell,the mechanism of this action may be related to the downregulation of N －cadherin,Vimentin,Snail,Slug and MMP9 protein expression.So it may serve as a promising target in the treatment of nasopharyngeal carcinoma.