氧化应激影响肝癌细胞糖异生关键基因表达的作用

Effects of Oxidative stress on the expression of key gluconeogenesis genes in hepatoma cells

目的:研究氧化应激在肝癌细胞(HepG2细胞)中对糖异生关键基因G6PC (glucose-6-phosphatase,G-6-pase)和GLUT4 (glucose transport protein 4)的影响,并揭示其潜在的调控机制。方法:利用双氧水(H2O2)处理肝癌细胞导致其细胞内氧化应激水平升高,然后通过qRT-PCR检测G6PC基因和GLUT4基因在不同时间和不同浓度药物处理后的转录表达情况,再通过Western Blot技术检测G6PC和GLUT4在不同时间和不同浓度药物处理后的蛋白表达变化。最后,通过免疫荧光实验对比双氧水处理前后G6PC和GLUT4的蛋白表达情况以及转录因子FOXO1(Forkhead box O 1)的蛋白定位情况,从而揭示氧化应激在肝癌细胞中调控糖异生的作用机制。结果:(1) qRT-PCR实验结果表明G6PC基因和GLUT4基因的转录表达与H2O2处理的时间和浓度均呈正相关;(2)Western Blot实验结果表明G6PC和GLUT4的蛋白表达与H2O2处理的时间和浓度均呈负相关;(3) H2O2处理后,免疫荧光结果显示G6PC和GLUT4蛋白整体荧光亮度较对照组减弱,但G6PC和GLUT4在核内的荧光亮度较对照组稍强;(4) H2O2处理后,免疫荧光结果显示转录因子FOXO1在核内的荧光亮度较对照组减弱及胞质荧光亮度较对照组增强。结论:氧化应激能够诱导转录因子FOXO1从细胞质转入细胞核,从而导致HepG2肝癌细胞糖异生基因(G6PC和GLUT4)的转录表达升高。

Objective: To study the effects of oxidative stress on G6PC (glucose-6-phosphatase G-6-pase) and GLUT4 (glucose transport protein4) in HepG2 hepatoma cells and further reveal its potential regulatory mechanism. Methods: Treatment of hepatoma cells with hydrogen peroxide (H 2O 2) resulted in the increased level of intracellular oxidative stress. Then, by qRT-PCR and Western Blot, we will detect the transcriptional expression or protein expression levels of G6PC and GLUT4 genes at different times and concentrations respectively. Finally, by using immunofluorescence experiments, we will determine the protein localization of the transcription factor FOXO1(Forkhead box O 1) and the protein expression of G6PC and GLUT4 before and after H 2O 2 treatment, thus revealing the potential regulatory mechanism of oxidative stress in gluconeogenesis of hepatoma cells. Results:(1) The results showed that the transcriptional expression of G6PC and GLUT4 genes were significantly positively correlated with the time and concentration of H 2O 2 treatment by qRT-PCR assays.(2) The results showed that the protein expression of G6PC and GLUT4 were significantly negatively correlated with the time and concentration of H 2O 2 treatment by Western Blot assays.(3) After treatment with H 2O 2, the results revealed that the overall fluorescence intensity of G6PC and GLUT4 protein were weaker than that of the control group, but the fluorescence intensity of G6PC and GLUT4 in the nucleus were slightly stronger than that of the control group by immunofluorescence assays.(4) After treatment with H 2O 2, immunofluorescence results revealed that the fluorescence intensity of the transcription factor FOXO1 in the nucleus was weaker than that of the control group, and the fluorescence intensity in the cytoplasm was stronger than that of the control group. Conclusions: Oxidative stress induce the transcription factor FOXO1 to relocalize from the cytoplasm to the nucleus, thus increasing the transcriptional expression of gluconeogenesis genes ( G6PC , GLUT4 ) in HepG2 hepatoma cells.