siRNA沉默LOX基因对鼻咽癌细胞侵袭及迁移能力的影响

The effect of down-regulating LOX on invasion and migration in nsopharyngeal carcinoma cells

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siRNALOX-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-cadherin、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 LOXdown - regulation on the migration and invasion of nasopharyngeal carcinoma cell line 5 - 8F in vitro. Metliods ： siRNA（ siRNA - LOX -01 and siRNA - LOX -02） that can transiently 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 were performed to examine cell metastasis. The expressions of LOX , E - cadherin , N - cadherin , Vimentin , Snail,Slug and MMP were determined by Western blot. Results ： Compared with cells in knocking down of LOX decreased transcription and expression in 5 - 8F cells showing by blot. The invasion and migration of the cells transfected with siRNA - LOX were much group （ P 〈 0. 05 ） . The expressions of LOX,N - cadherin,Vimentin,Snail, Slug and MMP 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 MMP protein expression. So it may serve as a promising target in the treatment of nasopharyngeal carcinoma.