摘要目的 探讨赖氨酰氧化酶(LOX)基因特异性RNA干扰对乳腺癌细胞侵袭转移的影响及机制.方法 构建LOX基因特异性的慢病毒干扰载体(LOX-RNAi-LV),转染至乳腺癌细胞MDA-MB-231,实时定量荧光PCR检测MDA-MB-231细胞中LOX及转移相关因子基质金属蛋白酶(MMP)2、9的表达,Western印迹检测LOX蛋白的表达,细胞侵袭试验(Transwell小室)检测转染前后细胞侵袭迁移能力变化.免疫组织化学法检测111例乳腺癌组织、癌旁乳腺组织及20例乳腺良性病变组织中LOX的表达,并对其与临床病理资料及MMP-2、MMP-9的相关性进行分析.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,抑制率分别为89.2％±1.3％和84.4％±0.4％;干扰组细胞MMP-2、MMP-9 mRNA相对表达量分别为0.496±0.021和0.571±0.099,均明显低于阴性对照组(0.846±0.047,0.786±0.042)和空白对照组(1.000±0.000,1.000±0.000)(均P＜0.05)；Transwell侵袭和迁移实验中,干扰组穿过Transwell小室滤过膜细胞数分别为47±2和63±2,均明显少于阴性对照组(100±1,118±2)和空白对照组(100±1,118±2)(均P=0.000)；LOX蛋白在人乳腺癌、癌旁乳腺组织及乳腺良性肿瘤组织中的表达率分别为48.6％(54/111)、26.1％(29/111)、20.0％(4/20),乳腺癌组织中LOX蛋白的表达率明显高于癌旁乳腺组织及乳腺良性肿瘤组织(P=0.019).LOX蛋白在不同肿瘤大小、临床分期、淋巴结转移的表达率差异有统计学意义.相关分析显示,LOX蛋白表达与MMP-2(r=0.262,P=0.005)、MMP-9(r=0.424,P=0.000)蛋白之间呈显著正相关.结论 LOX可以促进乳腺癌的侵袭转移；LOX和MMP-2、MMP-9转移因子可能具有协同促进作用,促进了乳腺癌的侵袭转移.

abstractsObjective To investigate possible mechanism of silencing lysyl oxidase (LOX) gene by RNA interference affecting on invasion and metastasis of breast cancer cells.Methods LOX-RNAi-LV was designed and synthesized,which was transfected into breast cancer cell line MDA-MB-231.The expressions of LOX,MMP-2 and MMP-9 were determined by Real-time PCR in MDA-MB-231 cells,and the protein expression of LOX was determined by Western blot.The cells migration and invasion abilities were measured by cell migration and invasion test.111 eases of breast cancer tissue and cancer-adjacent breast tissues and 20 cases of benign lesion tissues of LOX,MMP-2 and MMP-9 were detected by immunohistochemistry,and the relationship of LOX and clinicopathological characteristics was analyzed.Results The expression levels of LOX mRNA and protein were down-regulated obviously after transfecting LOX-RNAi-LV,with the inhibition rate 89.2％±1.3％ and 84.4％±0.4％ repectively.The relative expressions of MMP-2 and MMP-9 mRNA were 0.496±0.021 and 0.571±0.099 in RNAi group,which was significantly lower than that in negative control group ( 0.846±0.047,0.786±0.042 ) and blank control group ( 1.000±0.000,1.000±0.000 ) ( both P＜0.05 ).Cell migration and invasion test showed the average cell numbers per field in the group RNAi were 47±2 and 63±2,was significantly lower than that in negative control group (100±1,118±2) and blank control group (100±1,118±2) (both P＜0.05).The expression of LOX protein in breast cancer,cancer-adjacent breast tissues and benign breast tumor were 48.6％ (54/111),26.1％ (29/111),20.0％ (4/20),the expression of LOX protein in breast cancer was significantly higher than that in cancer-adjacent breast tissues and benign lesion tissues (P=0.019).The expression of LOX protein was associated with clinical stage,lymph node metastasis,tumor size.Correlation analysis showed that LOX protein expression was significantly positive correlation with MMP-2 (r=0.262,P=0.005) and MMP-9 (r=0.424,P=0.000).Conclusion LOX can promote invasion and metastasis of breast cancer;LOX and MMP-2,MMP-9 may have a synergistic role in promoting invasion and metastasis of breast cancer.