摘要
目的有关RNF19A在肺癌中的生物学功能尚不清楚。文中旨在探讨RNF19A对肺癌细胞增殖、侵袭、迁移能力的影响及机制。方法实验根据肺癌A549细胞处理不同分为对照组(阴性随机序列,无干扰作用)和RNF19A干扰组(敲低肺癌A549中RNF19A的表达),采用CCK8法检测细胞活力,BrdU掺入实验检测细胞增殖,检测RNF19A对A549细胞相关信号通路的影响,利用免疫共沉淀技术和蛋白免疫印迹法检测RNF19A与TAK1蛋白相互作用对TAK1泛素化水平的影响,TAK1抑制剂及NF-κB抑制剂对Flag-RNF19A介导的A549细胞增殖、迁移、侵袭的影响。结果 CCK8及BrdU掺入实验结果显示,RNF19A干扰组48h时A549细胞增殖能力(0.62±0.04)较对照组(0.83±0.04)明显降低,72 h的结果亦明显降低(P<0.001)。细胞迁移和侵袭实验结果表明,RNF19A干扰组细胞的迁移数、侵袭数[(441.0±18.63)个、(488.2±26.06)个]明显低于对照组[(960.6±37.82)个、(1120±58.96)个],差异有统计学意义(P<0.05)。RNF19A干扰组细胞内TAK1的泛素化水平较对照组明显降低[(0.425±0.01)vs(0.656±0.012),P<0.05]。过表达Flag-RNF19A可以促进A549细胞的增殖(P<0.05)。与对照质粒比较,Flag-RNF19A迁移数、侵袭数[(1032±38.86)个、(657.7±13.74)个]较对照质粒[(721.7±26.60)个、(355.7±15.51)个]明显增加(P<0.05);但加入TAK1抑制剂及NF-κB抑制剂后,过表达Flag-RNF19A促进A549细胞增殖的效应消失(P>0.05)。结论 RNF19A可以增强A549肺癌细胞的增殖、迁移和侵袭,其机制可能与增加TAK1泛素化水平和增强NF-κB活化有关。
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.
作者
薛伟
朱江波
XUE Wei;ZHU Jiang-bo(Department of Health Toxicology,Faculty of Naval Medicine,Second Militery Medical University,Shanghai 200433,China)
出处
《医学研究生学报》
CAS
北大核心
2019年第8期821-827,共7页
Journal of Medical Postgraduates
