小干扰RNA干扰β连环素表达对肺腺癌A549细胞Wnt信号通路的作用  

Effect of siRNA-mediated β-catenin gene on Wnt signal pathway in lung adenocarcinoma A549 cell

作　　者：滕颖[1] 王秀问[1] 王亚伟[1] 王健[1] 

机构地区：[1]山东大学齐鲁医院肿瘤中心化疗科,济南250012

出　　处：《中华医学杂志》2010年第14期988-992,共5页National Medical Journal of China

基　　金：山东省科学技术发展计划项目（No.2009GG20002044）

摘　　要：目的研究小干扰RNA（siRNA）干扰β连环素（β-catenin）表达对肺腺癌A549细胞Wnt／β-catenin信号通路的影响。方法将ASd9细胞分为转染β-catenin干扰质粒PGCsil—CTNNB1-siRNA组（干扰质粒组）、转染阴性对照质粒PGCsil-NC—siRNA组（阴性对照组）和未转染质粒组（空白对照组），对各组细胞用实时PCR法检测β-catenin及Wnt／β-catenin信号通路靶基因细胞周期蛋白（cyclin）D1mRNA的表达，以噻唑蓝法检测细胞增殖能力，流式细胞术进行细胞周期分析，克隆形成试验检测克隆形成率，Millicell小室试验检测细胞迁徙能力。结果干扰质粒组细胞β-cateninmRNA和cyclinD1mRNA表达均明显低于阴性对照组（0．002±0．001比0．023±0．002，P〈0．01；0．005±0．002比0．040±0．020，P〈0．05）和空白对照组（B-cateninmRNA：0．021±0．003，P〈0．01；cyclinD1mRNA：0．042±0．004，P〈0．05）；第5～7天细胞增殖能力明显弱于阴性对照组和空白对照组（P〈0．05，P〈0．01），细胞倍增时间（58．1h）明显长于阴性对照组（37．9h，P〈0．05）和空白对照组（34．2h，P〈0.05）；G0—G1期细胞（86．4％±2．6％）明显多于阴性对照组（73．8％±0．9％，P〈0．01）和空白对照组（75．8％±1．5％，P〈0．01）；细胞克隆形成率（31．6％±7．7％）明显低于阴性对照组（46．9％±7．3％，P〈0．05）和空白对照组（44．2％±2．5％，P〈0．05），穿过Millicell小室底膜的细胞数（16．0±3．8）明显低于阴性对照组（32．7±3．1，P〈0．01）和空白对照组（33．0±2．7，P〈0．01）。结论用siRNA干扰β—catenin的表达可抑制肺腺癌细胞Wnt／β-catenin信号通路，降低细胞的增殖能力、克隆形成能力和迁徙能力。Wnt／β-catenin信号通路可能成为肺癌分子靶向治疗的新靶点。Objective To study the effects of siRNA-mediated β-catenin down-regulation on Wnt/β-catenin signal transduction pathway in lung adenocarcinoma A549 cells. Methods A549 cell was divided into three groups： PGCsil-CTNNBI-siRNA group （transfection with β-catenin interference plasmids）, negative control group （transfection with negative control plasmids） and non-transfection group （blank control）. For each group, real-time PCR was employed to detect the expression of β-catenin and cyclin D1 （a target gene of Wnt/β-catenin pathway）. A M3T cell proliferation assay was performed to study the proliferating capacity. Flow cytometry was used for cell cycle analysis. Clone formation and Millicell chamber experiment were performed to evaluate the clone formation and migration capacities of cells. Results The expression of β-catenin and cyclin D1 in PGCsil-CTNNBI-siRNA cell decreased at the mRNA level. It was lower than negative control （ 0. 002 ± 0. 001 vs 0. 023 ± 0. 002, P 〈 0. 01 ; 0. 005 ± 0. 002 vs 0. 040 ± 0.020, P 〈0.05） and blank control groups （β-catenin mRNA： 0.021 ±0.003, P 〈0.01; cyclin D1 mRNA： 0. 042 ± 0. 004, P 〈 0.05 ） . Also the proliferating capacity at Days 5 - 7 was inhibited in comparison with negative control and blank control groups （ P 〈 0. 05, P 〈 0. 01 ）. The cell-doubling time （58.1 h） was markedly longer than those of negative control group （37. 9 h, P 〈0. 05） and blank control group （34. 2 h, P 〈 0. 05）. The number of cells in G0-G1 phrase increased in PGCsil-CTNNBI-siRNA group （ 86. 4% ± 2. 6% ） in comparison with negative control （ 73.8% ± 0. 9% , P 〈 0. 01 ） and blank control groups （75.8% ± 1.5% , P 〈0. 01 ）. In PGCsil-CTNNBI-siRNA group, the clone formation ratio （31.6% ±7. 7% ） was lower than negative control （46. 9% ±7.3% , P 〈0. 05） and blank control groups （44. 2% ±2. 5% , P 〈 0. 05 ）. And the number of cells （ 16. 0 ± 3. 8 ） passing through the membrane of Millicell chamb

关 键 词：肺肿瘤 腺癌 RNA 小分子干扰 Β连环素 信号传导 

分 类 号：R734.2[医药卫生—肿瘤]