ABSTRACT
Objective The biological function of E3 ubiquitin-protein ligase RNF19A in lung cancer is yet clear. This study was to investigate the effects of RNF19A on the proliferation, invasion and migration of lung cancer cells and its underlying mechanisms. Methods A549 cells were transfected with control siRNA or RNF19A siRNA for 48 hours. Then, the viability and proliferation of the cells were measured by CCK8 and BrdU incorporation assay, respectively. Immunoprecipitation and Western immunoblotting were used to detect the effect of RNF19A-TAK1 interaction on the ubiquitination of TAK1 and the effects of TAK1 and NF-κB inhibitors on the proliferation, invasion and migration of the Flag-RNF19A-mediated A549 cells. Results After 48 hours of transfection, the viability and proliferation of the A549 cells were significantly decreased in the RNF19A siRNA group as compared with the control group (P < 0.001), and so were the numbers of migrating (441.0 ± 18.63 vs 960.6 ± 37.82, P < 0.05) and invading cells (488.2 ± 26.06 vs 1120 ± 58.96, P < 0.05) and the level of TAK1 ubiquitination in the A549 cells (0.425 ± 0.01 vs 0.656 ± 0.012, P < 0.05). Over-expressed Flag-RNF19A markedly enhanced the proliferation of the cells in comparison with that of the control group (P < 0.05), and increased the numbers of migrating (1032 ± 38.86 vs 721.7 ± 26.60, P < 0.05) and invading cells (657.7 ± 13.74 vs 355.7 ± 15.51, P < 0.05), but showed no statistically significantly difference from the control in the proliferation of the cells with the addition of TAK1 and NF-κB inhibitors (P > 0.05). Conclusion RNF19A can increasing the proliferation, migration and invasion of A549 lung cancer cells, probably by enhancing TAK1 ubiquitination and NF-κB activation.
ABSTRACT
<p><b>OBJECTIVE</b>To establish the integrated discrete multiple organ cell culture (IdMOC) system.</p><p><b>METHODS</b>Rat primary cell of hepatocyte, nephrocyte, cardiomyocytes, alveolar macrophage, dermal fibroblasts were isolated by collagenase digestion, separation of bronchial lavage, two-step digestion method and cultured respectively, with monolayer culture. To establish the integrated discrete multiple organ cell culture (IdMOC) system, glass slides of five different cells were used to the same dish with 10% FBS DMEM medium cultured 7d, using MTT comparison primary cells cultured alone and cocultured when growth.</p><p><b>RESULTS</b>Established rat hepatocytes, renal cell, cardiomyocyte, alveolar macrophages, dermal fibroblasts separation method was stable, cell separation survival rate was about 90.0%. Hepatocytes separation survival rate 90.3% ,renal cell separation survival rate 91.9%, cardiomyocyte separation survival rate 93.0% and beating rate indifference curve among 3d-15d, alveolar macrophages cell separation survival rate 90.8%, dermal fibroblasts cell separation survival rate 92.7%. Five primary cells multiple organ cells coculture showed cocultured cell growth proliferation well, cultured alone and cocultured cells growth curve basic coincide.</p><p><b>CONCLUSION</b>Established rat multiple organ cell co-culture is successful.</p>