肝脏特异性敲除核因子-κB必需调节蛋白基因对Myc过表达诱导的小鼠原发性肝癌发生发展的影响

Effect of liver knockout of nuclear factor-κB essential modulator on the development of mouse hepatocarcinogenesis induced by Myc overexpression

目的:探讨IκB激酶(IKK)/核因子-κB(NF-κB)信号通路对Myc过表达诱导的小鼠原发性肝癌发生发展的影响。方法:利用转基因小鼠将LAP-tTA小鼠与Alb-cre小鼠杂交,再与NEMO f1(NEMO为NF-κB必需调节蛋白)小鼠杂交,以产生LAP-tTA+/Alb-cre+/NEMO fl/wt小鼠,最后与tetO-Myc+小鼠杂交。本实验包括4组:(1)WT小鼠;(2)NEMOΔLPC小鼠(肝细胞中NEMO敲除,无Myc过表达);(3)Myc LAP-tTA小鼠(Myc过表达,无NEMO敲除);(4)Myc LAP-tTA/NEMOΔLPC小鼠(肝脏Myc过表达和NEMO敲除)。记录小鼠的生存曲线,观察肝脏大体形态。苏木精-伊红(HE)染色观察肝组织病理结构,免疫组织化学染色、蛋白质印迹法(Western blot)检测肝脏肿瘤指标及肝祖细胞指标、定量聚合酶链反应(qPCR)方法检测相关蛋白的mRNA水平。使用t检验比较独立样本组与相应组。结果:小鼠带瘤生存曲线显示Myc LAP-tTA/NEMOΔLPC小鼠中位生存期明显短于Myc LAP-tTA小鼠(中位生存期M/P 5056 d比83 d,P<0.01);Myc LAP-tTA/NEMOΔLPC小鼠对比Myc LAP-tTA小鼠,谷草转氨酶[(739.40±35.61)U/L比(441.50±78.79)U/L,t=2.464,P<0.05]、碱性磷酸酶[(3142.0±287.6)U/L比(1702.0±278.8)U/L,t=3.129,P<0.01]、γ-谷氨酰基转移酶[(101.70±12.82)U/L比(37.31±9.34)U/L,t=3.927,P<0.01]和总胆红素[(1.281±0.232)mg/dl比(0.618±0.051)mg/dl,t=3.889,P<0.01]均显著升高,且差异有统计学意义;Western blot检测显示细胞角蛋白19(CK19)、性别决定区Y框蛋白9(SOX9)水平在Myc LAP-tTA/NEMOΔLPC较Myc LAP-tTA组小鼠肝脏亦明显升高;肝祖细胞标志物如角蛋白(CK19,4.336±1.970比0.680±0.234,t=1.843,P<0.01)、CK7(3.884±0.338比2.370±0.525,t=2.422,P<0.01)、甲胎蛋白(AFP,3614.0±2070.0比1399.0±319.9,t=1.057,P<0.01)、上皮细胞黏附分子(EpCAM)的mRNA水平在Myc LAP-tTA/NEMOΔLPC较Myc LAP-tTA组小鼠肝脏显著增高(7.900±0.997比3.084±0.711,t=3.943,P<0.01)。结论:肝细胞中特异性敲除NEMO从而抑制经典的IKK/NF-κB信号通路,可促进肝肿瘤的发生发展,并诱导混合型肝细胞癌-胆管细胞癌的发生。白细胞介素(IL)-6/信号转导与转录激活因子-3(STAT3)通路与细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)通路可能参与混合型肝癌的发生发展。

Objective To investigate the role of IκB kinase(IKK)/nuclear factor-κB(NF-κB)signaling pathway in the development of Myc-induced mouse hepatocellular carcinoma and potential mechanisms.Methods Transgenic mice were used in the study.LAP-tTA mice were crossed with Alb-cre transgenic mice,and the double transgenic offsprings were crossed to nuclear factor-κB essential modulator(NEMOf1)mice to generate LAP-tTA+/Alb-cre+/NEMOfl/wt mice,which lastly were crossed to tetO-Myc+mice.Four groups of animals were included:(1)WT mice(no expression of Myc and no NEMO ablation),(2)NEMOΔLPC mice(specific deletion of NEMO in liver parenchymal cells but no expression of Myc),(3)MycLAP-tTA mice(only overexpression of Myc,no ablation of NEMO),and(4)MycLAP-tTA/NEMOΔLPC mice(both overexpression of Myc and ablation of NEMO).Kaplan-Meier survival curve was recorded,and liver was observed both macroscopically and microscopically.Hematoxylin and eosin(HE)staining was used to observe the pathology of liver tissues.Immunohistochemical staining,Western blotting and quantitative polymerase chain reaction(qPCR)were performed to detect liver tumor markers and liver progenitor cell markers.Results The Kaplan-Meier survival curve of mice showed that the median survival time of MycLAP-tTA/NEMOΔLPC mice was significantly shorter than that of MycLAP-tTA mice,and the difference was statistically significant(Median survival time M/P50,56 d vs.83 d,P<0.05).The serum levels of aspartate aminotransferase[(739.40±35.61)U/L vs.(441.50±78.79)U/L,t=2.464,P<0.05],alkaline phosphatase[(3142.0±287.6)U/L vs.(1702.0±278.8)U/L,t=3.129,P<0.01],γ-glutamin transferase[(101.70±12.82)U/L vs.(37.31±9.34)U/L,t=3.927,P<0.01]and total bilirubin[(1.281±0.232)mg/dl vs.(0.618±0.051)mg/dl,t=3.889,P<0.01]were significantly increased in livers of MycLAP-tTA/NEMOΔLPC mice compared to MycLAP-tTA mice(P<0.05);Western blotting analysis showed that cytokeratin 19(CK19)and sex determining region Y box 9(SOX9)protein levels in MycLAP-tTA/NEMOΔLPC mice were significantly higher than those in MycLAP-tTA group.The mRNA levels of hepatic progenitor cell markers such as keratin(CK19,4.336±1.970 vs.0.680±0.234,t=1.843,P<0.01),CK7(3.884±0.338 vs.2.370±0.525,t=2.422,P<0.01),alpha fetal protein(AFP,3614.0±2070.0 vs.1399.0±319.9,t=1.057,P<0.01)and epithelial cell adhesion molecule(EpCAM,7.900±0.997 vs.3.084±0.711,t=3.943,P<0.01)were significantly increased in MycLAP-tTA/NEMOΔLPC mice compared to MycLAP-tTA group(P<0.05).Conclusion Inhibition of IKK/NF-κB signaling pathway could accelerate Myc-induced hepatocarcinogenesis and induce the development of hepatocellular-cholangiocarcinoma(HCC-ICC).Interleukin(IL)-6/signal transducer and activators of transcription 3(STAT3)pathway and extracellular signal-regulated kinase(ERK)/mitogen-activated protein kinase(MAPK)pathway maybe involved in the progression of mixed HCC-ICC.