siRNA抑制人舌癌细胞VEGF表达对MMP-2、MMP-9及TIMP-1蛋白表达的影响

EFFECTS OF VEGF SUPPRESSION BY SIRNA ON THE EXPRESSION OF MMP-2,MMP-9 AND TIMP-1 IN HUMAN TONGUE SQUAMOUS CELL CAICINOMA

目的:探讨小干扰RNA(small interfering RNA,siRNA)抑制人舌癌Tca8113细胞血管内皮生长因子(vascular endo-thelial growth factor,VEGF)表达对移植瘤基质金属蛋白酶(matrix metalloproteinases,MMP)-2、MMP-9及基质金属蛋白酶抑制剂-1(tissue inhibitors of metalloproteinase-1,TIMP-1)蛋白表达的影响。方法:以稳定转染携带针对VEGF的siRNA真核表达载体的人舌癌Tca8113细胞(VEGF-siRNA1、VEGF-siRNA2)为实验组,以转染空载体的人舌癌Tca8113细胞及未转染的人舌癌Tca8113细胞为实验对照组和空白对照组,将各组细胞分别接种于裸鼠皮下,用明胶酶谱实验及蛋白免疫印迹法分别测定各组移植瘤TIMP-2、TIMP-9及TIMP-1的蛋白表达。结果:各组明胶酶谱实验结果均有较明显MMP-9蛋白表达,活性MMP-9蛋白含量较少,MMP-2蛋白表达量明显少于MMP-9蛋白表达,而活性MMP-2几乎完全没有可见条带出现。各组中MMP-2、MMP-9及活性-MMP-9条带酶量差异均尚不具统计学意义(P>0.05)。以scion图像分析系统分别读取各样本TIMP-1条带灰度值及β-actin条带灰度值,各组中TIMP-1蛋白相对含量差异亦无统计学意义(P>0.05)。结论:以siRNA干扰人舌癌Tca8113细胞VEGF基因,可明显抑制肿瘤内VEGF的蛋白表达,但是对MMP-2、MMP-9及TIMP-1蛋白的表达无明显影响。

Objective： To investigate the effects of vascular endothelial growth factor （VEGF） suppression by vector-based small interfering RNA （siRNA） on the expression of matrix metalloproteinase （MMP）-2, MMP-9 and tissue inhibitors of metalloproteinase-1 （TIMP-1） in human tongue squamous cell carcinoma （Tca8113） in vitro and in vivo. Methods： The eukaryotic expression vector was taken as an experimental control into human tongue squamous cell carcinoma cell lines （Tca8113）. The non-transfected Tca8113 cells were used as negative control. The transfected cells and non-transfected cells were injected subcutaneously on the back of twenty nudemice which were divided into four groups randomly （ n = 5/group） ; re spectively. The expression of MMP-2, MMP-9 and TIMP-1 in xenograft tumors was detected by Gelatin zymography and Western blot protein immunoblotting. Results： Compared with the experimental and negative controls, the VEGF expression was decreased （ P 〈0.05） in two experimental groups of the xenograft tumors. There was no significant difference in the expression of MMP-2, MMP-9 and TIMP-1 among four groups of xenograft tumor （ P 〉0. 05）. Conclusion： VEGF-siRNA can down-regulate the expression of VEGF in the human tongue squamous cell carcinoma in vitro and in vivo. However, it may not affect the expression of MMP-2, MMP-9 and TIMP-1 efficiently