PP2A-B56γ高表达抑制镉诱导的人肝L02细胞DNA损伤

doi:10.3969/j.issn.1004-616x.2012.03.006

›› 2012, Vol. 24 ›› Issue (3): 189-194.doi: 10.3969/j.issn.1004-616x.2012.03.006

PP2A-B56γ高表达抑制镉诱导的人肝L02细胞DNA损伤

林丽娜,林育纯,陈慧峰,罗洁,李晓杰,胡耀明,李文,张树江,陈雯,林忠宁

中山大学公共卫生学院，广东省营养膳食与健康重点实验室，广东广州 510080

收稿日期:2011-04-28 修回日期:2012-02-10 出版日期:2012-05-30 发布日期:2012-05-30
通讯作者: 林忠宁

Overexpression of PP2A-B56γ repressed the effect of cadmium on DNA damage in human normal L02 liver cells

LIN Li-na，LIN Yu-chun，CHEN Hui-feng，LUO Jie，LI Xiao-jie，HU Yao-ming，LI Wen，ZHANG Shu-jiang，CHEN Wen，LIN Zhong-ning

School of Public Health, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Guangzhou 510080, Guangdong, China

Received:2011-04-28 Revised:2012-02-10 Online:2012-05-30 Published:2012-05-30
Contact: LIN Zhong-ning

摘要/Abstract

摘要： 目的：探讨人正常肝细胞内蛋白磷酸酶2A-B56γ亚基 (PP2A-B56γ，由PPP2R5C基因编码)高表达对镉诱导相关基因转录水平和DNA损伤效应的影响，并探讨其作用机制。方法：构建PP2A-B56γ高表达L02-2R5Cc和空白载体对照L02-pBabe细胞模型；两种细胞分别给予CdCl2 (Cd)、TNFα单独处理及联合处理后，实时荧光定量-PCR(QRT-PCR)检测不同处理组PPP2R5C和金属硫蛋白1B (MT1B) mRNA转录水平；彗星试验(SCGE)检测细胞DNA断裂损伤情况；并按照3×2的析因设计分析不同处理之间是否存在联合作用及作用类型。结果：与L02-pBabe细胞相比较，L02-2R5Cc细胞中PPP2R5C、外源性PPP2R5C-FLAG mRNA显著高表达 (P<0.05)，细胞株构建成功。3种因素单独处理时，与阴性对照组相比，Cd降低PPP2R5C、升高MT1B mRNA表达，PPP2R5C基因高表达诱导的效应与Cd处理相反，TNFα降低PPP2R5C和MT1B mRNA表达 (均P<0.05)；析因分析表明，在PPP2R5C和PPP2R5C-FLAG mRNA检测中，PPP2R5C高表达与Cd、与TNFα之间均存在协同性交互效应 (P<0.05)；在MT1B mRNA检测中，PPP2R5C高表达与Cd和TNFα之间均表现为拮抗作用，PPP2R5C高表达与TNFα为协同抑制作用 (均P<0.05)。SCGE检测表明，与阴性对照相比，Cd诱导彗星尾长、尾矩、尾部DNA含量和拖尾细胞阳性率显著增高 (P<0.05)，TNFα、PPP2R5C高表达单独作用均不引起DNA损伤 (P>0.05)，但两两联合作用的析因分析结果表明，TNFα预处理与Cd处理对DNA损伤具有协同作用，PPP2R5C高表达与Cd处理存在拮抗作用，交互作用均具有统计学意义 (P<0.05)。结论：Cd抑制肝细胞PPP2A-B56γ编码基因的转录并显著诱导DNA损伤，PP2A-B56γ高表达抑制镉诱导的DNA损伤，而炎症因子可增强镉对细胞DNA的损伤。

关键词: 蛋白磷酸酶2A (PP2A), 调节亚基B56γ, 镉, 肝细胞毒性, 基因转录调控

Abstract: OBJECTIVE: To investigate the effects of protein phosphatase 2A (PP2A)-B56γ subunit (encoded by PPP2R5C gene) overexpression on cadmiun-induced gene transcription and DNA damage in human normal liver L02 cells. METHODS：Cell models were established via stable transfection with overexpression PPP2R5C (L02-2R5Cc) and null vector pBabe-puro (L02-pBabe). QRT-PCR was used to detect the effects of cadmium and TNFα on PPP2R5C and MT1B mRNA expression. SCGE assay was adopted to evaluate their genotoxicity. The 3 × 2 factorial analysis experiment was aimed to explore the type of their combined effects. RESULTS：The levels of total and exogenous (PPP2R5C-FLAG) PPP2R5C mRNA were markedly elevated in L02-2R5Cc cells compared with L02-pBabe cells，and reduced by treatments with cadmium and TNFα (P<0.05). The level of MT1B mRNA was induced by cadmium but reduced by TNFα and PPP2R5C overexpression (P<0.05). Factorial ANOVA analysis revealed that，in terms of the mRNA levels of PPP2R5C and PPP2R5C-FLAG，synergistic enhanced effects were showed between PPP2R5C overexpression and cadmium treatment，also PPP2R5C overexpression and TNFα treatment (P<0.05). While on MT1B mRNA expression，there was antagonistic effect between PPP2R5C overexpression and cadmium as well as between cadmium and TNFα，but synergistic inhibitory effect between PPP2R5C overexpression and TNFα (P<0.05). DNA damage was significantly induced by cadmium (P<0.05)，but not detected by TNFα nor PPP2R5C overexpression (P > 0.05). Further factorial analysis suggested synergistic effects were found between cadmium and TNFα，but antagonistic effect between cadmium and PPP2R5C overexpression (P<0.05). No interaction was noted between PPP2R5C overexpression and TNFα (P> 0.05). CONCLUSION：Cadmium suppressed PPP2R5C gene transcription and significantly induced DNA damage. PP2A-B56γ overexpression repressed the genotoxicity induced by cadmium，which was enhanced in the presence of inflammatory factor.

Key words: protein phosphatase 2A (PP2A), regulatory B56γ subunit, cadmium, hepatotoxicity, transcription regulation

中图分类号:

R735.7

林丽娜,林育纯,陈慧峰,罗洁,李晓杰,胡耀明,李文,张树江,陈雯,林忠宁. PP2A-B56γ高表达抑制镉诱导的人肝L02细胞DNA损伤[J]. , 2012, 24(3): 189-194.

LIN Li-na，LIN Yu-chun，CHEN Hui-feng，LUO Jie，LI Xiao-jie，HU Yao-ming，LI Wen，ZHANG Shu-jiang，CHEN Wen，LIN Zhong-ning. Overexpression of PP2A-B56γ repressed the effect of cadmium on DNA damage in human normal L02 liver cells[J]. , 2012, 24(3): 189-194.

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PP2A-B56γ高表达抑制镉诱导的人肝L02细胞DNA损伤

Overexpression of PP2A-B56γ repressed the effect of cadmium on DNA damage in human normal L02 liver cells

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