低氧对Jurkat细胞低氧诱导因子-1α及其类泛素化的影响及意义

Effect of hypoxia on HIF-1αand its sumoylation in Jurkat cells

目的 探讨低氧条件下Jurkat细胞低氧诱导因子-1α（HIF-1α）类泛素化的变化对其基因转录活性和蛋白稳定性的影响,以及对下游靶基因转录活性调控的影响和意义.方法 氯化钴（CoCl2）化学缺氧法模拟肿瘤缺氧不同时间,分别用荧光定量PCR、Western blot法检测HIF-1α基因及蛋白稳定性的变化,类泛素-1（SUMO-1）、类泛素蛋酶-1（SENP-1）蛋白水平的表达,VEGF、mdr1、mdr3、Mcl-1和survivin等基因转录活性的变化.结果 在缺氧诱导4、8、16、48、72 h后,HIF-1α基因转录活性分别为缺氧诱导前的（0.79 ±0.19）、（2.65±2.05）、（4.19±4.72）、（2.77±3.37）、（0.09±0.05）、（0.69±0.55）倍（P〉0.01）,而蛋白稳定性逐渐增高后减低（P〈0.01）.SEN-1蛋白的表达与SUMO-1蛋白表达呈相反趋势,前者逐渐增高后减低,而后者逐渐减低后增高（P〈0.01）.除survivin 基因外,VEGF、mdr1、mdr3、Mcl-1基因转录水平与HIF-1α蛋白稳定性相平行.结论 缺氧引起SENP-1表达变化,通过减低HIF-1α蛋白类泛素化作用而影响HIF-1α稳定性,从而改变下游VEGF、mdr1、mdr3、Mcl-1等基因的转录活性而最终影响细胞牛物学过程.

Objective To investigate the effects of hypoxia on sumoylation of HIF-1α,as well as transcriptional activity and protein stability of HIF-1α in Jurkat cells,and explore its effect and significance on modulating the transcriptional activity of down stream target gene.Methods CoCl2 was used as a chemical inducer to simulate the hypoxia environment.Real-time fluorescence quantitative PCR and western blot were used to evaluate transcriptional activity and protein stability of HIF-1α.levels of SUMO-1 and SENP1 protein,and gene transcripts of VEGF,mdr1,mdr3,Mcl-1 and survivin respectively.Results After 4 h,8h,16 h,24 h and 72 h after hypoxia induction,the gene transcripts of HIF-1α were 0.79±0.19,2.65±2.05,4.19 4-4.72,2.77±3.37,0.09±0.05 and 0.69±0.55-fold（P〉0.01） of that of Oh in Jurkat cells,respectively,while the protein stability increased first,then decreased（P〈0.01）.SENP-1 protein upregulated first,then down-regulated,and the SUMO-1 protein changed in an opposite trend.Excepting for survivin gene,transcriptional activities of VEGF,mdr1,mdr3,and Mel-1 were affected by the stability of HIF-1α protein.Conclusion Hypoxia induces changes in SENP-1 expression,which increases the stability of HIF-1α by decreasing the sumoylation of HIF-1α and affects biological process by regulating the transeriptional activities of VEGF,mdr1,mdr3 and Mcl-1 gene.