高铜通过转录水平上调LC3表达诱导肝癌HepG2细胞自噬的研究  

High copper induces autophagy of HepG2 cells by up-regulating LC3 expression at transcriptional level

作　　者：张萌[1] 石萌 南欣睿 张杰[1] 杜文倩 李超[1] 刘宝琴[1] ZHANG Meng;SHI Meng;NAN Xin-rui;ZHANG Jie;DU Wen-qian;LI Chao;LIU Bao-qin(School of Life Sciences,China Medical University,Shenyang l10122,China)

机构地区：[1]中国医科大学生命科学学院,辽宁沈阳110122

出　　处：《中华肿瘤防治杂志》2022年第24期1734-1740,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81872257);辽宁省自然科学基金(2022-MS-199);省博士科研启动基金计划(2020-BS-101)。

摘　　要：目的探究微量元素铜对肝癌HepG2细胞自噬水平的影响,解析铜离子诱导肝癌自噬的分子机制。方法0、10、50、100和200μmol/L硫酸铜(CuSO_(4))处理HepG2细胞24 h,100μmol/L CuSO_(4)处理HepG2细胞0、4、8、12、24 h后检测自噬相关蛋白表达,蛋白质印迹法检测HepG2细胞内自噬分子标志物LC3-Ⅱ/LC3-Ⅰ和p62,以及线粒体自噬标志分子Parkin表达;实时荧光定量PCR(qRT-PCR)检测LC3Ⅱ和p62 mRNA表达,细胞计数试剂盒检测细胞活力。RFP-GFP荧光双标LC3慢病毒系统感染HepG2细胞,CuSO_(4)处理表达RFP-GFP-LC3B的HepG2细胞,共聚焦检测自噬流变化。结果10、50、100、200μmol/L CuSO_(4)作用HepG2细胞后,LC3-Ⅱ/LC3-Ⅰ比值呈剂量(F=6519.000,P<0.001)和时间依赖性升高(F=934.300,P<0.001);p62蛋白表达呈剂量(F=55564.000,P<0.001)和时间依赖性降低(F=11413.000,P<0.001)。共聚焦结果显示铜离子蓄积诱导HepG2细胞内自噬小体数目(t=30.040,P<0.001)及自噬溶酶体数目(t=272.000,P<0.001)均增加。100μmol/L CuSO_(4)引起HepG2细胞中LC3-Ⅱ/LC3-Ⅰ比值增加(t=103.600,P<0.001);p62蛋白表达下降(t=146.600,P<0.001),EBSS也引起p62蛋白表达下降(t=146.000,P<0.001);而对Parkin蛋白表达没有影响。100μmol/L CuSO_(4)作用HepG2细胞24 h后LC3ⅡmRNA表达上调,而EBSS饥饿处理则不影响LC3ⅡmRNA表达,F=302.400,P<0.001;p62 mRNA表达均一定程度上调,F=61.750,P<0.001。细胞计数实验结果表明100μmol/L CuSO_(4)和EBSS饥饿均使细胞活力降低,F=111.700,P<0.001。结论高铜能够通过转录水平上调LC3Ⅱ分子mRNA表达诱导肝癌HepG2细胞内自噬增强。Objective To investigate the effect of trace element copper on autophagy of HepG2 cells,and to elucidate the molecular mechanism of copper-induced autophagy of HepG2 cells.Methods Copper sulfate(CuSO_(4))at concentration of 0.10,50,100 and 200μmol/L was used to treat HepG2 cells for 24 h;after the HepG2 cells were treated with 100 mol/L CuSO_(4)for 0,4,8,12 and 24 hours,the expression of autophagy-related proteins was detected.The expression of autophagy molecular markers LC3-Ⅱ/LC3-Ⅰand p62 in HepG2 cells and the expression of mitochondrial autophagy marker Parkin were detected by western blot,The expression of LC3Ⅱand p62 mRNA was detected by real-time fluorescent quantitative PCR(qRT-PCR),and the cell viability was detected by cell counting kit.HepG2 cells were infected by RFPGFP fluorescent double labeled LC3 lentivirus system.HepG2 cells expressing RFP-GFP-LC3B were treated with CuSO_(4),and autophagic flow changes were detected by confocal detection.Results After the HepG2 cells were treated with 10,50,100 and 200μmol/L CuSO_(4),the LC3-Ⅱ/LC3-Ⅰratio increased in a dose-dependent manner(F=6519.000,P<0.001)and time-dependent manner(F=934.300,P<0.001).The expression of p62 protein decreased in a dose-dependent manner(F=55564.000,P<0.001)and time-dependent manner(F=11413.000,P<0.001).The confocal results showed that the number of autophagosomes(t=30.040,P<0.001)and autophagic lysosomes(t=272.000,P<0.001)in HepG2 cells induced by copper ion accumulation increased.CuSO_(4)at a concentration of 100μmol/L increased the LC3-Ⅱ/LC3-Ⅰratio in HepG2 cells(t=103.600,P<0.001).The expression of p62 protein decreased(t=146.600,P<0.001),and EBSS also caused the decrease of p62 protein expression(t=146.000,P<0.001).It had no effect on Parkin protein expression.After 24 hours of exposure to 100μmol/L CuSO_(4),the expression of LC3ⅡmRNA in HepG2 cells was up-regulated,while EBSS starvation did not affect the expression of LC3ⅡmRNA(F=302.400,P<0.001).The expression of p62 mRNA was up-regulated to a certain e

关 键 词：肝癌 铜稳态 LC3 自噬 P62 

分 类 号：R735.7[医药卫生—肿瘤]