CAJ | 학술논문

目的探讨缺氧对肝癌细胞内β-链蛋白(β-catenin)表达的影响及机制.方法分别利用100 μmol/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)、Western blot、免疫荧光和免疫组织化学法分析缺氧对PLC/PRF/5、Hep3B、HepG2和MHCC97 4种肝癌细胞内β-catenin mRNA和蛋白表达的影响.结果缺氧不影响肝癌细胞β-catenin mRNA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.
목적 탐토결양대간암세포내β-련단백(β-catenin)표체적영향급궤제.방법 분별이용100 μmol/L록화고화간암원위이식+간동맥결찰건립간암세포체내、외결양모형.실시정량역전록-취합매련반응(RT-PCR)、Western blot、면역형광화면역조직화학법분석결양대PLC/PRF/5、Hep3B、HepG2화MHCC97 4충간암세포내β-catenin mRNA화단백표체적영향.결과 결양불영향간암세포β-catenin mRNA표체,단증가HepG2화PLC/PRF/5세포내β-catenin총단백수평(＞2.3배),촉진MHCC97급Hep3B세포β-catenin적세포내역위(포질화/혹포핵,＞1.7배).저여결양유도적간암세포내당원합성매3β하조,β-catenin단백강해억제유관.결론 결양촉진간암세포내β-catenin단백표체화세포내전이,증가세포악성잠능.
Objective To investigate the effect of hypoxia on expression of β-catenin in hepatocellular 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 CoCl2 ) 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 cells.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-3β) and proteasome inhibiting assay.Results Hypoxia induced a pronounced increase in total β-catenin levels in PLC/PRF/5 and HepG2 cells ( ＞ 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 xenograft 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 downregulation 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 metastatic potential.