人核因子κBp65核定位信号缺失突变对A549细胞恶性表型的影响

The Influence of Human NF-κBp65 NLS Deletion Mutant Plasmids on Malignant Phenotype of A549 Lung Cancer Cells

目的鉴定人核因子κB(NF-κB)p65核定位信号(NLS)缺失突变质粒即pc DNA3.1(+)-NF-κBp65ΔNLS(简称NF-κBp65ΔNLS)的表达功能,以及其对低表达NF-κBp65的A549细胞株即A549/NF-κBp65 shRNA细胞增殖、迁移和粘附能力的影响。方法培养人A549/NF-κBp65 shRNA细胞株,分为对照组、转染pc DNA3.1(+)组、转染NF-κBp65ΔNLS组。应用间接免疫荧光、实时荧光定量-PCR和Western blot技术检测NF-κBp65的细胞内定位及mRNA、蛋白表达水平的变化;应用MTT、Transwell和细胞粘附实验分析转染NF-κBp65ΔNLS质粒对A549/NF-κBp65 shRNA细胞增殖、迁移、粘附能力的影响。结果成功构建人NF-κBp65ΔNLS真核表达质粒。转染NF-κBp65ΔNLS质粒的A549/NF-κBp65 shRNA细胞与对照组及转染pc DNA3.1(+)组比较,NF-κBp65 mRNA表达水平明显上调(10.63±0.84比1.04±0.21和1.23±0.22,P<0.01),NF-κBp65蛋白表达水平明显升高(1.07±0.06比0.53±0.02和0.59±0.04,P<0.01),且NF-κBp65蛋白主要位于细胞浆内,在肿瘤坏死因子α刺激后NF-κBp65蛋白并未明显进入细胞核。与对照组及转染pc DNA3.1(+)组比较,转染NF-κBp65ΔNLS质粒的A549/NF-κBp65 shRNA细胞的增殖、迁移和粘附能力均有不同程度增强。结论通过基因突变技术构建无NLS的NF-κBp65真核表达质粒,可明显增强A549/NF-κBp65shRNA细胞株内NF-κBp65 mRNA和蛋白的表达水平,并使NF-κBp65蛋白定位于细胞浆内;同时,细胞浆内过表达NF-κBp65ΔNLS蛋白可明显增强A549/NF-κBp65 shRNA细胞的增殖、迁移和粘附能力,提示滞留于细胞浆内的NF-κBp65仍可通过影响NF-κB信号通路相关蛋白参与调节肺癌细胞的生物学行为。

Objective To identify the expression functions of human NF-κBp65 nuclear localization signals' deletion mutant plasmids( namely pc DNA3. 1( +)-NF-κBp65ΔNLS,NF-κBp65ΔNLS,for short)and the changes of proliferation,migration and adhesion ability of A549 lung cancer cells with low expression of NF-κBp65( namely A549 / NF-κBp65 shRNA cells). Methods Human A549 / NF-κBp65 shRNA cells were cultivated and divided into a control group,a transfection pc DNA3. 1( +) group,and a transfection NF-κBp65ΔNLS group. Indirect immunofluorescence,real-time fluorescent quantitative PCR and Western blot techniques were used to detect the NF-κBp65 intracellular localization and the change of NF-κBp65mRNA and protein expression level. MTT,Transwell and cell adhesion experiments were used to analyze the changes of proliferation,migration and adhesion ability of A549 / NF-κBp65 shRNA cells. Results The human NF-κBp65ΔNLS eukaryotic expression plasmid was successfully constructed. Compared with the control group and the transfection pc DNA3. 1( +) group,NF-κBp65 mRNA expression level in A549 /NF-κBp65 shRNA cells was increased in the transfection NF-κBp65ΔNLS group( 10. 63 ± 0. 84 vs. 1. 04 ±0. 21 and 1. 23 ± 0. 22,P < 0. 01) and NF-κBp65 protein expression level was also increased( 1. 07 ± 0. 06 vs. 0. 53 ± 0. 02 and 0. 59 ± 0. 04,P < 0. 01). NF-κBp65 protein mainly located in the cytoplasm,and did not significantly transferred into the nucleus after stimulated by TNF-α. At the same time,A549 / NF-κBp65shRNA cells' proliferation,migration and adhesion ability were enhanced compared with the control group and the transfection pc DNA3. 1( +) group. Conclusions Through gene mutation technology to build the human NF-κBp65ΔNLS eukaryotic expression plasmid and transfect into A549 / NF-κBp65 shRNA lung cancer cell lines,both mRNA and protein expression levels of NF-κBp65 were increased significantly,and NF-κBp65 protein mainly located in the cytoplasm. The overexpressed NF-κBp65 in cytoplasm can obviously enhance the A549 / NF-κBp65 shRNA cell's proliferation,migration and adhesion ability. It suggests that NF-κBp65 stranded in the cytoplasm can still regulate biological behavior of lung cancer cells by influencing the NF-κB signaling pathway related proteins.