缺氧对肝癌细胞内β-链蛋白表达的影响

The diverse changes of β-catenin induced by hypoxia in hepatocellular carcinoma

目的探讨缺氧对肝癌细胞内β-链蛋白（13-catenin）表达的影响及机制。方法分别利用100μmol／L氯化钴和肝癌原位移植＋肝动脉结扎建立肝癌细胞体内、外缺氧模型。实时定量逆转录．聚合酶链反应（RT—PCR）、Westernblot、免疫荧光和免疫组织化学法分析缺氧对PLC／PRF／5、Hep3B、HepG2和MHCC974种肝癌细胞内B—cateninmRNA和蛋白表达的影响。结果缺氧不影响肝癌细胞β-cateninmRNA表达，但增加HepG2和PLC／PRF／5细胞内β-catenin总蛋白水平（〉2．3倍），促进MHCC97及Hep3B细胞β-catenin的细胞内易位（胞质和／或胞核，〉1．7倍）。这与缺氧诱导的肝癌细胞内糖原合成酶3β下调，β-catenin蛋白降解抑制有关。结论缺氧促进肝癌细胞内β-catenin蛋白表达和细胞内转移，增加细胞恶性潜能。

Objective To investigate the effect of hypoxia on expression of β-catenin in hepatocel- lular carcinoma （HCC） and its molecular background. Methods Human HCC cell lines with different metastatic potentials （PLC/PRF/5, Hep3B, HepG2 and MHCC97 ） were grown under hypoxic （induced by 100 μmol/L CoC12 ） and normoxic conditions, respectively. The alterations of β-catenin due to hypoxia were examined by quantitative reverse transcription-polymerase chain reaction （RT-PCR） , Western blotting and immunofluensence staining in above ceils. The results in vitro were confirmed in the in vivo hypoxic model by using hepatic arterial ligation （HAL）. Its underlying mechanisms were investigated by analyzing the expression of glycogen synthase kinase 3β （GSK-3 β） and proteasome inhibiting assay. Results Hypoxia induced a pronounced increase in total β-catenin levels in PLC/PRF/5 and HepG2 ceils （ 〉 2. 3- fold） , but not in Hep3B and MHCC97 cells. The latter exhibited a marked intracellular translocation of β-catenin （ 〉 1.7-fold）, but no change in its total amount （ 〈 1.1-fold）. These results were confirmed by subcellular fractionation assays and immunofluoresence staining. In the in vivo MHCC97 and HepG2 xeno-graft models, Western blotting and immunostaining showed that HAL increased β-catenin expression in HepG2 xenografts （ 〈 1.3-fold）, compared to its sham-operated controls, while intracellular translocation was achieved in MHCC97 xenografts. The elevated β-catenin level in HCC cells was attributed to downreg- ulation of GSK-3β expression and proteasome inhibition. Conclusion Hypoxia promotes overexpression and intracellular accumulation of β-catenin in HCC cells, which was correlated with hypoxia-induced meta-static potential.