急性缺血缺氧诱导肝癌细胞自噬及其相关机制

Induction of autophagy by ischemia-hypoxia in hepatocellular carcinoma

目的:研究急性缺血/缺氧时肝癌细胞HepG2增殖率及自噬的变化,探讨自噬的作用及机制。方法 :以Western印迹法检测缺氧诱导因子-1(hypoxia induced factor-1,HIF-1)表达并确定体外模拟模型的可靠性;吖啶橙染色后采用荧光显微镜定性观察自噬;以自噬特异性蛋白LC3及P62(P62/SQSTM1)信号蛋白的变化表明自噬的诱导和可能的调控机制;以CCK8检测3-甲基腺嘌呤(3-MA)抑制自噬前后急性缺血/缺氧下HepG2细胞的增殖率变化。结果:急性缺血/缺氧2 h后,HepG2细胞显著表达HIF-1α蛋白,表明体外急性缺血/缺氧模型的可靠性。急性缺血/缺氧可快速诱导肝癌细胞HepG2产生自噬,继而出现HepG2细胞显著增殖活跃;3-MA抑制自噬后,可特异性抑制急性缺血/缺氧所诱导的HepG2细胞增殖;急性缺血/缺氧条件下HepG2细胞P62信号蛋白表达显著下调,自噬抑制后逐渐恢复正常表达水平。结论:自噬在肝癌体外急性缺血/缺氧过程中对肿瘤起到保护作用,其机制可能与P62蛋白的清除有关;抑制自噬可显著降低肝癌细胞增殖率。自噬可能成为肝癌治疗的新靶点。

Objective To investigate the role and mechanism of autophagy on the cell viability of human hepatoma cell line HepG2 under acute ischemia/hypoxia. Methods Expression of hypoxia induced factor-1 （HIF-1） was deter- mined by Western blot to ensure the reliability of our in vitro cell model of acute ischemia/hypoxia induction. Quantitative analysis of autophagy was performed by fluorescent microscope on acridine orange staining. The changes in cell viability induced by acute ischemia/hypoxia before and after autophagy inhibitor 3-methyl adenine （3-MA） treatment were analyzed using cell counting kit-8. The expressions of LC3 and P62 （P62/SQSTM1） proteins were estimated by Western blot. Results The significant increase of HIF-la after treatment confirmed the stability of acute ischemia/hypoxia simulation model in vitro. Acute ischemia/hypoxia induced autophagy in hepatoma cell line HepG2 significantly and afterwards led to active proliferation of cells. Inhibition of autophagy by 3-MA specifically led to a pronounced decrease in the proliferation of HepG2. Expression of P62 protein decreased significantly in HepG2 cells under acute ischemia/hypoxia induction and returned gradually to normal level after inhibition of autophagy. Conclusions Autophagy functions as a protector for he- patocellular carcinoma cell survival during acute ischemia/hypoxia. The mechanism involved may be due to the clearance of P62 protein. Autophagy inhibition can lower the viability of hepatocellular carcinoma cell. Autophagy can become a novel target therapy for hepatocellular carcinoma.