干扰LOX表达对MDA-MB-231细胞侵袭迁移影响及其机制的探讨

Influence of LOX gene expression on the invasion and migration of breast cancer cell line MDA-MB-231

目的:研究干扰赖氨酰氧化酶(LOX)基因表达对乳腺癌细胞MDA-MB-231体外侵袭迁移能力的影响,并探讨相关机制及意义。方法:设计合成特异性慢病毒干扰载体(LOX-RNAi-LV)转染MDA-MB-231细胞,实时定量PCR检测LOX mRNA和侵袭转移相关因子MMP-2、MMP-9和HIF-1α的mRNA表达,蛋白质印迹法检测LOX蛋白表达,细胞侵袭迁移实验(Transwell)检测细胞侵袭迁移能力。结果:转染LOX-RNAi-LV后,乳腺癌MDA-MB-231细胞LOXmRNA和蛋白表达水平明显降低,与空白组相比,抑制率分别为89.2%和82.97%。MMP-2(F=225.271,P=0.0000)、MMP-9(F=35.373,P=0.000 01)和低氧诱导因子HIF-1α(F=341.081,P=0.0000)的mRNA表达均明显降低。细胞侵袭迁移实验显示,干扰组穿过Transwell小室滤过膜的细胞数明显少于对照组(侵袭实验F=269.557,P=0.000 5;迁移实验F=164.321,P=0.000 3)。结论:转染LOX-RNAi-LV能有效地干扰LOX在乳腺癌细胞MDA-MB-231中表达,抑制肿瘤细胞的侵袭和迁移能力,其作用机制可能与下调MMP-2、MMP-9和HIF-1α表达有关。

OBJECTIVE： To investigate the influence of RNAi against lysyl oxidase （LOX） gene on invasion and migration of human breast cancer MDA-MB-231 cells in vitro. METHODS： Lentiviral interference vector （LOX-RNAiq.V） was designed and chemically synthesized, and stably transfected into human breast cancer cell line MDA-MPr231. The ex- pressions of LOX mRNA, MMP-2 mRNA, MMP-9 mRNA and HIF-lα mRNA were detected by Real Time PCR, and the expression of LOX protein was detected by Western blot analysis. The abilities of migration and invasion were ob served by cell invasion and motility test （Transwell） assay. RESULTS： Transfection of LOX-RNAi-LV into MDA-MB- 231 cells inhibited LOX gene expression and both of mRNA and protein expression levels markedly declined compared with blank group. The inhibition rate were 89.2 % and 82.97 %. LOX-RNAi-LV transfected MDA-MB-231 cells showed the expressions of MMP-2 mRNA （F=225. 271,P=0. 000 0）, MMP-9 mRNA （F=35. 373,P=0. 000 01） and HIF-la mRNA （F= 341.081, P = 0. 000 0） were decreased and the ability of invasion and motility was significantly decreased （invasion.. F= 269.557, P = 0. 000 5 ; motility ： F = 164.321, P = 0. 000 3 ）. CONCLUSIONS ： Transfection of LOX-RNAi-I.V could significantly interfere the expression of LOX and suppress the motility and invasion of MDA-MB-231 cells, which might be related with down-regulation of the expression levels of MMP-2, MMP-9 and HIF-lα.