常氧酸性环境对肝癌细胞HepG2 HIF-1α蛋白表达及其活性的影响

Effect of normoxic acidosis on HIF-1α protein expression and activity in human liver cancer cell line HepG2

目的:观察常氧酸性环境对体外肝癌细胞低氧诱导因子(hypoxia-inducible factor,HIF)-1α蛋白表达及其活性的影响,初步探讨其在肿瘤生长中的作用。方法:体外培养肝癌细胞HepG2,以MTT法测定常氧酸性环境(AP组,pH6.5)作用20h后细胞存活率,并与正常培养细胞(SD组,pH7.2)作比较;Western印迹法检测AP组和SD组HepG2细胞核内HIF-1α蛋白表达的变化;应用转录因子DNA结合ELISA试剂盒(TransAMTM)分析AP组和SD组HepG2细胞核内HIF-1DNA结合活性的变化;免疫细胞化学法和Western印迹法检测各组HepG2细胞内血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白表达的变化。结果:常氧酸性环境(pH6.5)作用20h后,AP组细胞存活率为(98.71±1.79)%,与SD组相比轻微降低,但无统计学意义(P>0.05)。Western印迹结果表明,AP组HepG2细胞核内HIF-1α和胞内VEGF蛋白条带平均灰度值分别是SD组的(9.34±1.67)倍和(3.42±0.83)倍,组间均有统计学差异(P<0.01)。AP组HepG2细胞核内HIF-1DNA结合活性明显高于对照组(P<0.01)。结论:常氧酸性环境能显著提高HepG2细胞HIF-1蛋白含量及其DNA结合活性,同时其下游基因VEGF蛋白含量增加,推测除低氧环境外,酸性环境也可能是导致恶性肿瘤中HIF-1高表达的原因之一。

Objective：To investigate the effect of normoxic acidosis on HIF-1α protein expression and activity in human liver cancer cell line HepG2,so as to assess the role of acidosis in tumor growth.Methods：HepG2 cells were cultured in vitro,the cell growth was assessed by MTT assay after exposure to normoxic acidosis culture media（AP,pH 6.5）for 20 h,and the results were compared with those of the control cells（SD,pH 7.2）.Nuclear extractions were performed in the cultured cells,and HIF-1α protein level and HIF-1 DNA binding activities were examined by immunocytochemistry,Western blotting,and HIF-1α Transcription Factor Assay Kit（TransAMTM）.The protein level of VEGF（vascular endothelial growth factor）was detected in whole cell extractions by Western blotting assay.Results：MTT assay demonstrated that the cell survival rate was（98.71±1.79）% after treatment with normoxic acidosis culture media（AP,pH 6.5）.In comparison with SD group,we noted a slight decrease of cell viability in AP group（P〉0.05）.Western blotting analysis showed that the levels of HIF-1α and VEGF protein increased by（9.34±1.67）folds and（3.42±0.83）folds,respectively（P〈0.01）.Moreover,normoxic acidosis greatly enhanced HIF-1 DNA-binding activity compared to SD groups（P〈0.01）.Conclusion：Normoxic acidosis can evidently increase HIF-1α protein level and DNA-binding activity in HepG2 cells.Furthermore,the protein level of VEGF,an important target gene of HIF-1,is also increased.Therefore it is suggested that,in addition to hypoxia,acidosis may also play an important role in induction of HIF-1 in cancer.