摘要
目的探究营养缺乏对人肝母细胞瘤HepG2细胞自噬流的影响及ATG5介导的对抗营养应激的自噬依赖性分子途径。方法将慢病毒转染到HepG2细胞,敲降ATG5基因的表达,通过Western blot和qRT-PCR双向验证转染情况,使用HBSS盐缓冲液饥饿诱导处于对数生长的人肝癌细胞HepG2细胞0、1、2、3、4、5、6、7 h后,采用单丹磺酰尸胺(monodansylcadaverine,MDC)荧光染色法检测细胞内自噬小体的形成;运用Western blot分析微管相关蛋白1A/1B-轻链3(microtubule-associated protein 1A/1B-light chain 3,LC3)、自噬受体蛋白(sequestosome 1,P62)、自噬相关基因5(autophagy-related gene 5,ATG5)和营养应激通路(AMPK/mTOR通路)的表达变化;用CCK-8检测细胞的增殖活性。结果与常规培养比较,饥饿处理HepG2细胞后,胞内MDC荧光颗粒数显著增加,饥饿6 h达峰值(P<0.0001),LC3B-Ⅱ、ATG5蛋白表达上升(P<0.05),P62蛋白表达下降(P<0.05),AMPK/mTOR通路随着饥饿时间增加逐渐被激活,自噬流强度增大;饥饿处理敲降ATG5的HepG2细胞,ATG5的表达降低(P<0.05),LC3B-Ⅱ与P62的变化无显著差异,自噬流强度受抑制,细胞增殖活性降低(P<0.0001)。结论人肝母细胞瘤HepG2细胞可以通过诱导ATG5表达调控自噬流对抗营养应激。
Objective To investigate the effect of nutritional deficiency on autophagic flux in human hepatoblastoma HepG2 cells and the autophagy-dependent molecular pathway mediated by ATG5 against nutritional stress.Methods Lentiviral siRNA knockdown of ATG5 was performed to knock down the expression in HepG2 cells,and the transfection was verified by Western blotting and qRT-PCR.HBSS was used to treat HepG2 cells for 0,1,2,3,4,5,6 and 7 h for starvation-induced autophagy.Monodansylcadaverine(MDC)fluorescence staining was employed to detect the formation of intracellular autophagic vesicles.Western blotting was performed to measure the expression changes in microtubuleassociated protein 1A/1B-light chain 3(LC3),autophagy receptor protein(sequestosome 1,P62),autophagyrelated gene 5(ATG5)and nutrient stress pathway(AMPK/mTOR pathway)related proteins.CCK-8 assay was utilized to test cell proliferation.Results Compared with the HepG2 cells under conventional culture medium,starvation induction resulted in more autophagic vacuoles(accumulation of autofluorescent marker,MDC),which peaked at 6 h of starvation(P<0.0001),up-regulated protein levels of LC3B-Ⅱand ATG5(P<0.05),decreased protein expression of P62(P<0.05),gradual activation of the AMPK/mTOR pathway with elapse of starvation time,and increase in the intensity of autophagic flux.However,in the HepG2 cells with ATG5 knockdown,starvation treatment led to decreased ATG5 expression,no significant difference in LC3B-II and P62 changes,inhibited autophagic flux,and suppressed cell proliferation(P<0.0001).Conclusion Human hepatoblastoma HepG2 cells can regulate autophagic flux to against nutritional stress by inducing ATG5 expression.
作者
赵莲
向冬云
吴惠仪
孙昊
邓敬桓
ZHAO Lian;XIANG Dongyun;WU Huiyi;SUN Hao;DENG Jinghuan(School of Public Health,Guangxi Medical University,Nanning,Guangxi,China;First School of Clinical Medicine,Guangxi Medical University,Nanning,Guangxi,China)
出处
《陆军军医大学学报》
北大核心
2025年第13期1454-1462,共9页
Journal of Army Medical University
基金
国家自然科学基金地区科学基金项目(82060434)
广西硕士研究生创新项目(YCSW2024260)。
