Background and objective Nuclear factor-κB is an important transcription factor and is closely associ-ated with a variety of malignant tumors. hTe biological behavior of lung tumor cells can be reversed by inhibiting the expres-sion of NF-κBp65 directly or indirectly. Nuclear factor-κBp65 gene shRNA recombinant plasmids were constructed and then infected with A549 cells. New stable cell lines were selected, and the ability of migration and adhesion was identiifed. Meth-ods Both scramble control sequence and interference sequence (shRNA) of human nuclear factor-κBp65 were designed and synthesized to build recombinant plasmids, with BamH I site at the 5′end and Xho I and EcoR I sites at the 3′end. A549 cells were infected, and stable transfection strains were selected by puromycin. Western blot and qRT-PCR methods were applied to assess the interference effcient of NF-κBp65 and the protein expression level of IκBα. Transwell and MTT assays were carried out to analyze the ability of migration and adhesion of A549 cells separately. Results Recombinant plasmids were successfully built, and A549/NF-κB p65 scramble and A549/NF-κB p65 shRNA stable transfection strains were also successfully screened. Both mRNA and protein expression levels of NF-κBp65 showed that A549/NF-κBp65 shRNA cells decreased compared with A549/NF-κB p65 scramble cells and A549 cells, whereas the protein level of IκBαsigniifcantly increased. Both migration and adhesion abilities were also reduced. Conclusion In this study, both mRNA and protein expression levels of NF-κBp65 were effectively suppressed by RNA interference technique. NF-κBp65 inhibition can signiifcantly reduce the migration and adhe-sion ability of A549 cells.%背景与目的核因子-κB作为重要转录因子，与多种恶性肿瘤的发生发展有着密切联系，直接或间接抑制NF-κBp65的表达可逆转肿瘤细胞生物学行为。构建人核因子-κBp65基因shRNA重组质粒，感染A549细胞并筛选出稳定细胞株，对其迁移、粘附能力进行鉴定。方法设计并合成人核因子-κBp65的乱序对照序列（scramble）和干扰序列（shRNA）构建重组质粒，在5’端引入一个BamHI位点，3’端引入一个XhoI位点和EcoRI位点；感染A549细胞并由嘌呤霉素做稳转株的筛选；应用Western blot、qRT-PCR技术检测NF-κBp65的干扰效果及IκBα蛋白表达水平的变化；Transwell、MTT法分析其对A549细胞迁移、粘附能力的影响。结果成功构建重组质粒并筛选出A549/NF-κB p65 scramble稳转株和A549/NF-κB p65 shRNA稳转株；A549/NF-κB p65 shRNA稳转细胞株与A549/NF-κB p65 scramble稳转细胞株及A549细胞相比NF-κBp65的mRNA、蛋白表达水平均下调；IκBα蛋白水平明显下调；细胞迁移能力及粘附能力均降低。结论本实验通过RNA干扰技术构建的重组慢病毒可有效抑制NF-κBp65 mRNA和蛋白的表达水平；抑制NF-κBp65可明显降低A549细胞的迁移和粘附能力。
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