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. 2014 May;17(5):369-77.
doi: 10.3779/j.issn.1009-3419.2014.05.02.

[Construction and influence of human nuclear factor-κB p65 shRNA lentiviral vector on malignant biological behavior of lung cancer cells]

[Article in Chinese]
Hongjuan Guo  1 , Guangfa Zhu  1 , Chunting Wu  1
Affiliations

[Construction and influence of human nuclear factor-κB p65 shRNA lentiviral vector on malignant biological behavior of lung cancer cells]

[Article in Chinese]
Hongjuan Guo et al. Zhongguo Fei Ai Za Zhi. 2014 May.

Abstract
in English, Chinese

Background and objective: Nuclear factor-κB is an important transcription factor and is closely associated with a variety of malignant tumors. The biological behavior of lung tumor cells can be reversed by inhibiting the expression 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 identified.

Methods: 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 efficient 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 IκBα significantly increased. Both migration and adhesion abilities were also reduced.

Conclusions: In this study, both mRNA and protein expression levels of NF-κBp65 were effectively suppressed by RNA interference technique. NF-κBp65 inhibition can significantly reduce the migration and adhesion 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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Figures

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NF-κBp65 shRNA慢病毒载体和NF-κBp65 scramble慢病毒载体酶切鉴定结果 Identified results of NF-κBp65 shRNA lentiviral vector and NF-κBp65 scramble lentiviral vector
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乱序对照序列和干扰序列测序结果（部分）。A: NF-κBp65 scramble测序结果；B: NF-κBp65 shRNA测序结果。 Sequencing results of scramble control sequences and interference sequences (partly). A: sequencing result of scramble control sequences; B: sequencing result of interference sequences.
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293T细胞对NF-κBp65 scramble和NF-κBp65 shRNA慢病毒载体的包装结果。a、b、c、d、e和f为转染24 h后293T细胞；g、h、i、j、k和l为转染48 h后293T细胞。其中a、b、c、g、h、i为自然光下293T细胞；d、e、f、j、k、l为红色荧光下293T细胞（bar=100 μm）。 Packaging results of NF-κBp65 scramble lentiviral vector and NF-κBp65 shRNA lentiviral vector by 293T cells. a, b, c, d, e and f are 293T cells that have been transfected 24 h; g, h, i, j, k and l are 293T cells that have been transfected 48 h; a, b, c, g, h and i are 293T cells under white light; d, e, f, j, k and l are 293T cells under red fluorescence (bar=100 μm).
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筛选出的A549/NF-κBp65 scramble稳转细胞株和A549/NF-κBp65 shRNA稳转细胞株。a、b和c为自然光下A549细胞；d、e和f为红色荧光下A549细胞（bar=50 μm）。 Stably transfected cell strains of A549/NF-κBp65 scramble and stably transfected cell strains of A549/NF-κBp65 shRNA that have been screened out. a, b and c are A549 cells under white light; d, e and f are A549 cells under red fluorescence (bar=50 μm).
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不同A549稳转细胞株中NF-κBp65的mRNA表达水平。转染NF-κBp65 shRNA后的A549细胞NF-κBp65 mRNA表达水平较对照组A549细胞和转染NF-κBp65 scramble后的A549细胞均明显降低；转染NF-κBp65 scramble后的A549细胞NF-κBp65 mRNA表达水平与对照组A549细胞无明显差异。a：A549细胞；b：A549/NF-κBp65 scramble细胞；c：A549/NF-κBp65 shRNA细胞。NS：P > 0.05；*P < 0.05；**P < 0.01。 Expression levels of NF-κBp65 mRNA in different stably transfected A549 cell strains Expression level of NF-κBp65 mRNA was significantly reduced in A549/NF-κBp65 shRNA cells than that in A549 cells and A549/NF-κBp65 scramble cells, but no difference between A549 cells and A549/NF-κBp65 scramble cells. a: A549 cell; b: A549/NF-κBp65 scramble cell; c: A549/NF-κBp65 shRNA cell; NS: P > 0.05; *P < 0.05; **P < 0.01.
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不同A549稳转细胞株中NF-κBp65蛋白和IκBα蛋白表达水平。A: A549/NF-κBp65 shRNA细胞及对照组A549细胞和A549/NF-κBp65 scramble细胞中NF-κBp65蛋白和IκBα蛋白的western blot结果，以GAPDH蛋白为内参；B:转染NF-κBp65 shRNA后的A549细胞NF-κBp65蛋白表达水平较对照组A549细胞和转染NF-κBp65 scramble后的A549细胞均明显降低；转染NF-κBp65 scramble后的A549细胞NF-κBp65蛋白表达水平与对照组A549细胞无明显差异；C:转染NF-κBp65 shRNA后的A549细胞IκBα蛋白表达水平较对照组A549细胞和转染NF-κBp65 scramble后的A549细胞均明显降低；转染NF-κBp65 scramble后的A549细胞IκBα蛋白表达水平与对照组A549细胞无明显差异。a：A549细胞；b：A549/NF-κBp65 scramble细胞；c：A549/NF-κBp65 shRNA细胞。NS：P > 0.05；*P < 0.05；**P < 0.01。 Expression levels of NF-κBp65 protein and IκBα protein in different stably transfected A549 cell strains. A: The western blot results of NF-κBp65 protein and IκBα protein in A549/NF-κBp65 shRNA cells, A549 cells and A549/NF-κBp65 scramble cells. GAPDH served as an internal control; B: Expression level of NF-κBp65 protein was significantly reduced in A549/NF-κBp65 shRNA cells than that in A549 cells and A549/NF-κBp65 scramble cells, but no difference between A549 cells and A549/NF-κBp65 scramble cells; C: Expression level of IκBα protein was significantly reduced in A549/NF-κBp65 shRNA cells than that in A549 cells and A549/NF-κBp65 scramble cells, but no difference between A549 cells and A549/NF-κBp65 scramble cells. a: A549 cell; b: A549/NF-κBp65 scramble cell; c: A549/NF-κBp65 shRNA cell. NS: P > 0.05; *P < 0.05; **P < 0.01.
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不同A549稳转细胞株间细胞迁移数、粘附率的比较。A、B、C:分别为A549细胞、A549/NF-κBp65 scramble细胞和A549/NF-κBp65 shRNA细胞经transwell板上小孔发生迁移细胞的代表性视野（200×, bar=100 μm）；D: A549/NF-κBp65 shRNA细胞的迁移能力明显低于A549细胞和A549/NF-κBp65 scramble细胞；E: A549/NF-κBp65 shRNA细胞的粘附率明显低于A549细胞和A549/NF-κBp65 scramble细胞。a：A549细胞；b：A549/NF-κBp65 scramble细胞；c：A549/NF-κBp65 shRNA细胞。NS：P > 0.05；*P < 0.05；**P < 0.01。 Comparisons about numbers of migrated cells and adhesion ratio among different stably transfected A549 cell strains. A, B and C showed representative fields of cells migrated through filters in transwell inserts from A549, A549/NF-κBp65 scramble and A549/NF-κBp65 shRNA cells respectively (200×, bar=100 μm); D: The ability of migration was significantly reduced in A549/NF-κBp65 shRNA cells than that in A549 cells and A549/NF-κBp65 scramble cells, but no difference between A549 cells and A549/NF-κBp65 scramble cells; E: The adhesion ratio of A549/NF-κBp65 shRNA cells was significantly reduced than that in A549 cells and A549/NF-κBp65 scramble cells, but no difference between A549 cells and A549/NF-κBp65 scramble cells. a: A549 cell; b: A549/NF-κBp65 scramble cell; c: A549/NF-κBp65 shRNA cell. NS: P > 0.05; *P < 0.05; **P < 0.01.
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