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

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CdCl_2(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(MTIB)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。

OBJECTIVE：To investigate the effects of protein phosphatase 2A（PP2A）-B56γsubunit （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 TNFαon 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 MTIB mRNA was induced by cadmium but reduced by TNFαand 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 TNFαtreatment（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 TNFαnor 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-B56γoverexpression repressed the genotoxicity induced by cadmium,which was enhanced in the presence of inflammatory factor.