特异性ILKsiRNA对膀胱癌EJ细胞侵袭转移增殖-第三军医大学学报.DOC

特异性ILK-siRNA对膀胱癌EJ细胞侵袭和转移的影响 朱军，高娟，李红彦,潘湘阳，陈俊霞 (400016重庆，重庆医科大学细胞生物学与遗传学教研室，分子医学与肿瘤研究中心) [摘要] 目的 设计合成有效的ILK-siRNA,并检测其对EJ细胞侵袭和迁移的影响。方法 构建针对ILK基因mRNA的2个特异性siRNA表达载体pGenSil-1 siRNA1、 pGensil-1 siRNA2和1个阴性对照载体pGenSil-1 siRNA3。稳定转染膀胱癌EJ细胞后，通过RT-PCR检测EJ细胞中ILK mRNA水平的表达，并通过Western blot和细胞免疫荧光检测ILK蛋白水平的表达；观察EJ细胞形态变化；检测EJ细胞迁移、黏附和侵袭能力及基质金属蛋白酶的变化。结果 重组质粒构建成功。干扰组细胞ILK mRNA及蛋白的表达分别较未转染组、转空质粒组和转阴性对照质粒组相比，呈显著性下降（P0.05）；HE染色结果显示，细胞铺展良好，核质比减小；转染干扰质粒的2个实验组细胞的黏附能力相对于对照组分别增加87%和76%，黏附能力显著升高，迁移及侵袭能力明显降低（P0.05）；与其他3个对照组相比,转染 pGenSil-1 siRNA1与pGensil-1 siRNA2质粒到EJ细胞后,MMP-2及MMP-9的表达水平明显降低（P0.05）。结论 成功构建的干扰质粒能抑制ILK基因及蛋白水平的表达，显著抑制细胞的生长、侵袭及迁移能力。 [关键词] ILK;siRNA；膀胱癌EJ细胞；侵袭；迁移 ILK Silencing by Small Interfering RNA Inhibits Metastasis and Invasion Behaviour of Human EJ Bladder Cancer Cell Zhu Jun ,Gao Juan,Li Hongyan,Pan Xiangyang, Chen Junxia（Department of Cell biology and Genetics Division, Molecular Medicine and Cancer Research Center，Chongqing Medical University, Chongqing, 400016 ,China); [Abstract] Objective To investigate the effect of the specific siRNA of ILK on the migration and invasion of human EJ bladder cancer cells.Methods Construct two specific siRNA expression vectors and one none homology control vector. Transfect the vectors into EJ cells. Detect the RNAi efficiency by RT-PCR, Western-blot and Immunofluorescence. Observe the morphous change, determine the adhesion,migration and invasion ability.Results Enzyme digestion and DNA sequencing verified the construction correctly. In the two specific siRNA treatment groups,the expression of ILK was obviously inhibited , the cells spread well,the nuclear mass ratio was lower than control groups,the cell adhesion ability increased, the cell migration and metastasis ability decreased,the expression of MMP2 and MMP9 obviously decreased. Conclusion The specific siRNA of ILK was successfully constructed,ILK expression was obviously inhibited,and cell migration and metastasis ability significantly decreased. [Key words] I