膀胱癌细胞中锌指蛋白A20对核转录因子κB抑制的机制探讨

Possible mechanism of zinc finger protein A20 on nuclear transcription factor kappaB inhibition in urinary bladder cancer cell

目的 为了探讨锌指蛋白A2 0在膀胱癌细胞中抑制核转录因子κB(NuclearTranscriptionFactorKappaB ,NF κB)作用的可能机制。方法 转染绿色荧光蛋白融合的A2 0质粒 (GreenFluorescentProteinfusedA2 0 ,GFP A2 0 )和A2 0显性失活突变体质粒 [GFP A2 0 (1- 36 8) ]入膀胱癌细胞T2 4 ,荧光显微镜观察质粒在细胞中表达情况 ,RT PCR检测肿瘤坏死因子受体I(Tu morNecrosisFactorReceptorⅠ ,TNFRⅠ )、核转录因子κB受体激活子配体 (ReceptorActivatorofNF κBLigand ,RANKL)、NF κB抑制蛋白ⅠκBα的mRNA表达变化。结果 转染 2 4h后 ,质粒在细胞中稳定表达 ,4 8h后表达继续增加。转染A2 0质粒或A2 0显性失活突变体质粒 2 4h及 4 8h后 ,TNFRⅠ和RANKLmRNA表达无明显变化 (P >0 .0 5 )。而在这两个时间点处 ,A2 0质粒转染可明显抑制细胞内ⅠκBαmRNA的表达 ;A2 0质粒处理组与未处理组之间、A2 0质粒转染组与显性失活突变体质粒转染组之间差异均有显著性 (P <0 .0 1)。结论 A2 0作用于TNFR的下游对NF κB起抑制作用 ,ⅠκBαmRNA可能作为NF κB报告基因受A2 0抑制。

Objective To explore the possible reason of NF-κB inhibition by A20 in urinary bladder cancer cell. Methods Green fluorescent protein fused A20(GFP-A20) plasmid and dominant-negative A20 [GFP-A20(1-368)] plasmid were transfected into bladder cancer cell line T24, their expressions in cancer cells were detected by fluorescence microscopy 24 hours and 48 hours later. Semi-quantitative RT-PCR was performed to study the change of TNFR-Ⅰ, RANKL, ⅠκBα mRNA levels. Results GFP-A20 and GFP-A20(1-368) plasmids were stably expressed in T24 cells 24 hours after transfection and increased furtherly at 48h. TNFR-I and RNAKL mRNA levels were not changed by A20(GFP-A20) and GFP-A20(1-368) plasmids 24h or 48h after transfection ( P >0.05). ⅠκBα was significantly inhibited in two time points after GFP-A20 transfection. However, ⅠκBα was not affected by GFP-A20(1-368) plasmid. The statistical analyses between GFP A20 group and untreated group, GFP-A20 group and GFP-A20(1-368) group by semi-quantitative assay were significantly different( P <0.01). Conclusion A20 acted at downstream of TNFR to produce NF-κB inhibition. ⅠκBα mRNA, a reporter gene of NF-κB, was inhibited by A20.