缺氧诱导的外泌体活化自噬促进肝癌进展

Hypoxia-induced exosomes promote hepatocellular carcinoma progression through autophagy activation

目的探讨缺氧诱导的外泌体对肝癌进展的调节作用及其机制。方法将Huh7细胞置于0.1%O_(2)培养箱培养24 h作为缺氧组,另设常氧组,提取外泌体,纳米追踪技术检测外泌体的浓度和粒径大小,免疫印迹检测HSP70、Alix、CD63的表达。将Huh7细胞分为对照组(PBS)、常氧外泌体组(20μg/ml常氧外泌体)、缺氧外泌体组(20μg/ml缺氧外泌体),实时无标记细胞分析仪(real-time cell analysis,RTCA)实验检测细胞增殖,划痕实验检测细胞迁移,免疫印记检测细胞PCNA、cyclin D1、Bax、E-cadherin、N-cadherin、Vimentin、Beclin1、LC3、p62的表达,激光共聚焦检测自噬流的改变。Huh7细胞分为对照组(PBS)、缺氧外泌体组(20μg/ml缺氧外泌体)、缺氧外泌体+3-MA组(5 mmol/L 3-甲基腺嘌呤+1 h后20μg/ml缺氧外泌体),检测细胞增殖、迁移和相关蛋白的表达。结果与常氧组相比,缺氧组外泌体浓度增加,但外泌体粒径的大小没有明显变化,缺氧组外泌体标记蛋白HSP70、Alix、CD63的表达增加(P<0.05)。与常氧外泌体组相比,缺氧外泌体组Huh7细胞增殖和迁移能力增强(均P<0.05),PCNA、cyclin D1、N-cadherin、Vimentin、Beclin1、LC3-Ⅱ的表达上调,Bax、E-cadherin和p62的表达下调(均P<0.05),自噬流增强(P<0.05)。与缺氧外泌体组相比,缺氧外泌体+3-MA组Huh7细胞增殖、迁移和上皮间质转化作用减弱(P<0.05),PCNA、cyclin D1、N-cadherin、Vimentin、Beclin1、LC3-Ⅱ的表达下调(P<0.05),Bax、E-cadherin和p62的表达上调(P<0.05)。结论缺氧诱导的外泌体可以参与细胞自噬的发生进而促进肝癌进展。

Objective To investigate the role of hypoxia-induced exosomes in the progression of hepatocellular carcinoma and its related mechanism.Methods Huh7 cells were incubated for 24 h in a 1%O 2 hypoxia incubator in hypoxia group,with a normoxia group as the control.Exosomes were isolated from the cells in two groups to measure their concentration and size by nanoparticle tracking analysis(NTA).The expression levels of exosome protein markers HSP70,Alix,and CD63 were detected using Western blot.Huh7 cells were divided into control group(PBS),normoxia exosome group(20μg/ml normoxia-induced exosomes),and hypoxia exosome group(20μg/ml hypoxia-induced exosomes).The cell proliferation was measured by a real-time cell analyzer(RTCA),and the cell migration was examined by cell scratch assay.The expression levels of PCNA,cyclin D1,Bax,E-cadherin,N-cadherin,Vimentin,Beclin1,LC3 and p62 were analyzed by Western blot.Autophagic flux was detected by confocal microscopy.The cultured Huh7 cells were divided into control group(PBS),hypoxic exosome group(20μg/ml hypoxia-induced exosomes),and hypoxic exosome+3-MA group(5 mmol/L 3-methyladenine and 1 h later,20μg/ml hypoxia-induced exosomes),and then the cell proliferation,the cell migration and the expression of related proteins were detected.Results Compared with normoxia group,the exosome concentration was increased in hypoxia group,but the size of exosomes was not significantly different between the two groups.The expression levels of exosomal marker proteins HSP70,Alix,and CD63 in hypoxia group were higher than those in normoxia group(all P<0.05).Compared with normoxia exosome group,the proliferation and migration activities of Huh7 cells were increased in hypoxic exosome group,the expression of PCNA,cyclin D1,N-cadherin,Vimentin,Beclin1 and LC3-Ⅱwas upregulated,the expression of Bax,E-cadherin,and p62 was downregulated,and the autophagic flux was enhanced(all P<0.05).Compared with hypoxic exosome group,the proliferation and migration activities,and the epithelial mesenchymal transformation of Huh7 cells were weakened,the expression levels of PCNA,cyclin D1,N-cadherin,Vimentin,Beclin1,and LC3-Ⅱwere downregulated,and the expression levels of Bax,E-cadherin,and p62 were upregulated in hypoxic exosome+3-MA group(all P<0.05).Conclusion Hypoxia-induced exosomes may involve in the autophagy,thus exacerbating the progression of liver cancer.