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目的研究含缬酪肽蛋白(VCP)对肝癌细胞磷酸化核因子kappa B抑制蛋白(P-IκB)和核因子kappa B(NF-κB)信号的影响。方法聚合酶链式反应(PCR)扩增VCP基因,并克隆于真核表达载体p CDNA3.1/myc-His(-)A,测序正确后将其转染Huh7肝癌细胞,转染30 h后裂解细胞,免疫印迹试验(Western blot)检测核因子kappa B抑制蛋白(IκB)、P-IκB的含量,萤火虫荧光素酶报告基因检测NF-κB的活性;并对VCP RNA干扰,检测对IκB、P-IκB的含量和NF-κB的活性的影响。结果成功构建真核表达载体p CDNA3.1/myc-His(-)A-VCP,转染Huh7肝癌细胞后,IκB含量无明显变化,然而P-IκB含量明显降低,NF-κB信号活性显著增强;而对VCP RNA干扰后,NF-κB信号的活性受到明显抑制。结论 VCP通过促进Huh7肝癌细胞P-IκB的降解进而活化NF-κB,在肝癌的发生过程中发挥重要作用,是用于肝癌治疗的重要潜在靶点。
Objective To investigate the effect of valproate peptide (VCP) on the phosphorylation of nuclear factor kappa B inhibitor (P-IκB) and nuclear factor kappa B (NF-κB) in hepatocellular carcinoma cells. Methods VCP gene was amplified by polymerase chain reaction (PCR) and cloned into eukaryotic expression vector pCDNA3.1 / myc-His (-) A. The recombinant plasmid was transfected into Huh7 hepatoma cells after being sequenced. After 30 h of transfection, The expression of nuclear factor kappa B inhibitor (IκB) and P-IκB was detected by Western blot and the activity of firefly luciferase reporter gene in detecting NF-κB. VCP RNA interference was used to detect the expression of IκB, P-IκB content and NF-κB activity. Results The recombinant plasmid pCDNA3.1 / myc-His (-) A-VCP was successfully constructed. The expression of IκB was not significantly changed in Huh7 hepatoma cells, but the content of P-IκB was significantly decreased and the activity of NF-κB was significantly increased ; While the VCP RNA interference, NF-κB signal activity was significantly inhibited. Conclusion VCP can activate NF-κB by deactivating P-IκB in Huh7 hepatocarcinoma cells and plays an important role in the development of hepatocellular carcinoma and is an important potential target for the treatment of hepatocellular carcinoma.