赖氨酰氧化酶与乳腺癌侵袭转移相关因子的研究

摘要

目的 探讨赖氨酰氧化酶(LOX)与乳腺癌侵袭转移相关因子基质金属蛋白酶(MMPs)及低氧诱导因子-1α(HIF-1α)的相关性以及LOX在乳腺癌侵袭转移中的可能机制.方法 构建LOX基因的特异性慢病毒干扰载体(LOX-RNAi-LV),稳定转染至乳腺癌细胞MDA-MB-231,实验设空白组、干扰组和阴性对照组,实时定量荧光PCR检测3组细胞中LOX、MMP-2、MMP-9和HIF-1α的表达,Western blot检测各组LOX蛋白的表达；构建乳腺癌细胞MDA-MB-231,MCF-7和LOX基因干扰的MDA-MB-231裸鼠移植瘤模型,6周后测定移植肿瘤的生长及转移情况,SP免疫组织化学技术检测移植瘤中LOX、MMP-2、MMP-9及HIF-1α蛋白的表达情况.结果 转染LOX-RNAi-LV后乳腺癌细胞MDA-MB-231中的LOX mRNA和蛋白被明显抑制,抑制率为89.20％和82.97％,差异有统计学意义(P＜0.05)；干扰组的MMP-2、MMP-9及HIF-1α表达与阴性对照组及空白组比较差异有统计学意义(P＜0.05)；6周后裸鼠的MDA-MB-231组LOX、MMP-2、MMP-9及HIF-1α蛋白表达水平明显高于LOX基因干扰组及MCF-7组,相关分析显示,LOX蛋白与MMP-2(r=0.696,P=0.000)、MMP-9(r=0.735,P=0.000)、HIF-1α(r=0.737,P=0.000)蛋白呈显著正相关.结论 LOX-RNAi-LV可有效下调乳腺癌细胞MDA-MB-231中LOX mRNA和蛋白表达水平；LOX促进乳腺癌侵袭转移的机制可能与MMP-2、MMP-9及HIF-1α的异常表达相关.%Objective To investigate correlation between the lysyl oxidase(LOX),breast cancer invasion,metastasis related factors,matrix metalloproteinases(MMPs) and hypoxia inducible factor-1alpha(HIF-1α).Methods Lysyl oxidase gene-specific lentiviral RNA interference vetor(LOX-RNAi-LV)was designed and synthesized,which was stably transfected into breast cancer cell line MDA-MB-231.The breast cancer cells were divided into three groups:the experimental group,the negative control group and the blank control group.Real-time quantitative PCR analysis of LOX mRNA,MMP-2 mRNA,MMP-9 mRNA and HIF-1α mRNA expression.Western blot analysis LOX protein expression;To build the breast cancer cell line MDA-MB-231,MCF-7 and LOX genes interfere with MDA-MB-231 nude mice xenograft model.After 6 weeks,measured the transplantation nude mice of tumor growth and metastasis,SP immunohistochemistry chemical detected in xenografts LOX,MMP-2,MMP-9 and HIF-1α protein expression.Results After transfected with LOX-RNAi-LV,LOX mRNA and LOX protein in breast cancer cells MDA-MB-231 was significantly inhibited,inhibition rates were 89.20％ and 82.97 ％,the difference was statistically significant(P＜0.05) ;After 6 weeks,MDAMB-231 group LOX,MMP-2,MMP-9 and HIF-1α protein expression level was significantly higher than that of LOX gene interference group and MCF-7 group.The correlation analysis showed LOX protein and MMP-2 (r =0.696,P =0.000),MMP-9 (r =0.735,P=0.000) of HIF-1α(r=0.737,P=0.000) protein was significantly positively correlated.Conclusion LOX-RNAi-LV can be effectively lowered breast cancercells MDA-MB-231 of LOX mRNA and protein expression levels;LOX promote breast cancer invasion and metastasis mechanism may be related to MMP-2,MMP-9 and HIF-1α abnormal expression.