低氧条件下喉癌Hep-2细胞Stat3和HIF-1α表达关系的研究

The relationship between the expressions Stat3 and HIF-1α in Hep-2 cells under hypoxia

目的探讨低氧条件下喉癌Hep-2细胞磷酸化信号转导子和转录激活子3(phosphorylation signal transducer and activator of transcription 3,p-Stat3)及低氧诱导因子1α(hypoxiainduciblefactor1α,HIF-1α)、VEGF的表达及Stat3反义寡核苷酸(antisense oligodenucleotides,AS-ODN)对其表达的影响。方法应用流式细胞术检测常氧和低氧不同时间Hep-2细胞p-Stat3、HIF-1α、VEGF蛋白表达,检测Stat3AS-ODN转染Hep-2细胞24h,再于低氧条件下培养6h后Hep-2细胞p-Stat3、HIF-1α、VEGF蛋白表达。应用MTT法检测不同浓度(100、200nmol/L)Stat3AS-ODN、Stat3正义寡核苷酸(sense oligodenucleotides,S-ODN)、脂质体作用于Hep-2细胞后的细胞存活率。结果与常氧培养相比较,低氧培养条件下Hep-2细胞p-Stat3表达的荧光指数(fluorescent index,FI)在3h即有增长,6h后稳定;HIF-1α及VEGF的FI值6h和12h后增长,p-Stat3表达的变化与HIF-1α变化呈明显正相关性(r=0.691,P<0.01)。低氧条件下Stat3AS-ODN转染Hep-2细胞后抑制p-Stat3表达,HIF-1α、VEGF的蛋白表达FI值也随之下降,Pearson相关分析显示其变化呈正相关性(r=0.912,P<0.01)。Stat3AS-ODN转染Hep-2细胞后MTT细胞存活率随Stat3AS-ODN浓度(100、200nmol/L)增加而下降,与Stat3S-ODN对照组和脂质体对照组比较差异均有统计学意义(P均<0.01)。结论喉癌Hep-2细胞中存在由低氧激活的Stat3信号转导,p-Stat3于HIF-1α及VEGF表达之前出现增长,阻断Stat3后低氧条件下HIF-1α表达降低,提示Stat3调节HIF-1α表达。

Objective To investigate the expressions of p-Stat3, HIF-1α and VEGF in Hep-2 cells under hypoxia, and the effect of Star3 antisense oligonucleotides （AS-ODN） on these expressions. Methods Expressions of p-Star3 ,HIF-1α and VEGF of Hep-2 cells that were with or without Stat3 AS-ODN transfection, at hypoxic and normoxic conditions, were detected by flow cytometry. The survival rate of Hep-2 cells transfected with Stat3 AS-ODN and control groups were analyzed by MTT assay. Results The FI of p-Star3 was up-regulated in 3h-hypoxia group, and it was stable after 6h at hypoxic condition. The FI of HIF- 1α and VEGF increased after 6h and 12h at hypoxic condition. There was positive correlation between the expression of p-Stat3 and HIF-lct （ r = 0. 691, P 〈 0. 01 ）. Compared to Stat3 S-ODN group and lipofectamine control group, the expressions of p- Stat3, HIF-1α and VEGF in Stat3 AS-ODN group at hypoxic condition were significantly decreased （ P 〈 0. 01 ）. Pearson corre- lation analysis showed the significant positive correlation between the expressions of p-Star3 and HIF-1α （r = 0. 912 ,P 〈 0. 01 ）. The survival rate of Hep-2 cell reduced with the concentration augmentation of Star3 AS-ODN （ 100nmol/L and 200nmol/L） , which were significantly different from the Star3 S-ODN group and lipofectamine control group （P 〈 0. 01 ）. Conclusions Hy- poxia can induce Stat3 activation in Hep-2 cells. The expression of p-Stat3 is earlier than that of HIF-1α and VEGF. BlockingStat3 can decrease the expression of HIF-1α under hypoxia,indicating Stat3 might modulate HIF-1α expression in Hep-2 ceils at the hypoxic condition.