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 observe the expression of glutamate (Glu) andγ-aminobutyric acid (GABA) in the retina of diabetic rats which were intervened later by insulin intensive therapy, and to investigate the mechanism of metabolic memory of hyperglycemia which induced the retina neuropathy in diabetic rats. Methods60 Brown Norway rats were randomly divided into normal control (NC) group, diabetes mellitus (DM) group (6 weeks at DM1, 12 weeks at DM2) and metabolic memory (MM) group, 15 rats in each group. Diabetes was induced by intraperitoneal injection of streptozocin. After 6 weeks, MM group was treated with insulin intensive therapy for 6 weeks. DM1 group was sacrificed at the end of 6 weeks and other groups were sacrificed at the end of 12 weeks. High performance liquid chromatography was used to detect the amount of Glu and GABA in the rat retina. Real-time polymerase chain reaction was applied to quantify the mRNA expressions of Glutamate decarboxylase (GAD). TdT mediated dUTP nick ending labelling was used to detect cell apoptosis. ResultsThe concentration of Glu (t=6.963), GABA (t=4.385) and the ratio of Glu/GABA (t=4.163) in MM group were significantly higher than DM1 group, but the concentration of Glu (t=3.411) and GABA (t=3.709) were significantly lower than DM2 group (P < 0.05). And there was no significant difference in the ratio of Glu/GABA between MM and DM2 groups (t=1.199, P > 0.05). The level of expressions of GAD mRNA in MM group was significantly lower than DM1 group (t=3.496, P < 0.05), but higher than DM2 group (t=8.613, P < 0.05). The number of nerve cells apoptosis in MM group was significantly higher than DM1 group (t=2.584, P < 0.05), but lower than DM2 group (t=3.531, P < 0.05). ConclusionsIntensive therapy later by insulin can partially reduce the content of Glu and GABA and the rate of nerve cells apoptosis, which cannot return to normal levels, and has no effect on the rise in the ratio of Glu/GABA caused by the hyperglycemia. The disorders of Glu and GABA may participate in the metabolic memory of hyperglycemia.