ObjectiveTo identify the expression functions of human NF-κBp65 nuclear localization signals' deletion mutant plasmids(namely pcDNA3.1(+)-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). MethodsHuman A549/NF-κBp65 shRNA cells were cultivated and divided into a control group, a transfection pcDNA3.1 (+) 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-κBp65 mRNA 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. ResultsThe human NF-κBp65ΔNLS eukaryotic expression plasmid was successfully constructed. Compared with the control group and the transfection pcDNA3.1(+) 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-κBp65 shRNA cells' proliferation, migration and adhesion ability were enhanced compared with the control group and the transfection pcDNA3.1(+) group. ConclusionsThrough 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.
ObjectiveTo study the correlation between international normalized ratio (INR) and coagulation factor Ⅱ and Ⅹ in patients with pulmonary thromboembolism treated with warfarin at moderate and low intensity anticoagulation.MethodsFifty-one patients with pulmonary thromboembolism treated with warfarin orally were divided into low-intensity anticoagulation group (INR from 1.6 to 2.0) and standard-intensity anticoagulation group (INR form 2.0 to 3.0) according to their monitoring INR indices. The levels of coagulation factor Ⅱ and Ⅹ were measured, and the correlation between INR level and coagulation factor activity was compared.ResultsThe INR of the low intensity anticoagulation group was 1.69±0.2 and the standard intensity anticoagulation group was 2.55±0.46. The corresponding activity of coagulation factor Ⅱ was (48.3±28.0)% and (24.0±8.0)% respectively. The activity of coagulation factor Ⅹ was (32.8±24.0)% and (16.7±6.0)%. There was a negative correlation between the activity of INR and coagulation factor Ⅱ and Ⅹ, with correlation coefficients of –0.903 and –0.459, respectively. Coagulation factor Ⅱ activity < 40%, coagulation factor Ⅹ activity inhibitory level < 25% is defined as anticoagulation effect. When coagulation factor Ⅱ activity level reaches anticoagulation effect, the corresponding minimum INR value was 1.56 and as to coagulation factor Ⅹ, the corresponding minimum INR value was 1.66.ConclusionsINR is negatively correlated with the activity of coagulation factor Ⅱ and coagulation factor Ⅹ. With the increase of INR, the activity of coagulation factor Ⅱ and coagulation factor Ⅹ decrease. Low intensity anticoagulation could not effectively inhibit the activity of coagulation factor.