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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 of NF-κB p65ΔNLS (NF-κBp65ΔNLS), which is a deletion mutant plasmid of NF-κB p65 nuclear localization signal (NLS), and its effect on the expression of NF-κB p65 A549 cell line A549 / NF-κBp65 shRNA proliferation, migration and adhesion. Methods Human A549 / NF-κBp65 shRNA cell line was cultured and divided into control group, pcDNA3.1 (+) transfected group and NF-κBp65ΔNLS group. The expression of NF-κBp65 was detected by indirect immunofluorescence, real-time fluorescence quantitative PCR and Western blot. The expression of NF-κBp65ΔNLS was detected by MTT assay, Transwell assay and cell adhesion assay. Effect of NF-κBp65 shRNA on Cell Proliferation, Migration and Adhesion. Results The human NF-κBp65ΔNLS eukaryotic expression plasmid was successfully constructed. The expression of NF-κBp65 mRNA was significantly up-regulated in A549 / NF-κBp65 shRNA transfected with NF-κBp65ΔNLS compared with that in control and pcDNA3.1 (+) group (10.63 ± 0.84 vs 1.04 ± 0.21 vs 1.23 ± 0.22 (P <0.01, P <0.01). The expression of NF-κBp65 protein was significantly increased (1.07 ± 0.06 vs. 0.53 ± 0.02 and 0.59 ± 0.04, P <0.01), and the NF-κBp65 protein mainly located in the cytoplasm. After NF-κBp65 protein did not significantly enter the nucleus. The proliferation, migration and adhesion of A549 / NF-κBp65 shRNA transfected with NF-κBp65ΔNLS plasmid were enhanced to some extent compared with the control group and transfected pcDNA3.1 (+) group. Conclusion The construction of a NLS-free NF-κBp65 eukaryotic expression plasmid by gene mutation can significantly enhance the expression of NF-κBp65 mRNA and protein in A549 / NF-κBp65shRNA cell lines and locate the NF-κBp65 protein in the cytoplasm. Meanwhile, overexpression of NF-κBp65ΔNLS in cytoplasm significantly enhanced the proliferation, migration and adhesion of A549 / NF-κBp65 shRNA cells, suggesting that NF-κBp65 retained in the cytoplasm may be related to NF-κB signaling pathway Proteins are involved in the regulation of the biological behavior of lung cancer cells.