过表达Survivin通过调控缺氧血管内皮细胞EPAS1和CHOP-10表达影响细胞增殖、凋亡

Overexpression of survivin affects cell proliferation and apoptosis by regulating the expression of EPAS1 and CHOP-10 in vascular endothelial cells with hypoxic treatment

摘要:

目的：研究过表达存活素（Survivin，SVV）基因对缺氧处理的大鼠血管内皮细胞增殖、迁移能力及内皮PAS1区域蛋白1（endothelial PAS domain-containing protein 1，EPAS1）、C/EBP同源蛋白-10（C/EBP homologous protein-10，CHOP-10）蛋白表达的影响和机制。方法：用500 μmol/L 氯化钴（CoCl2）缺氧处理大鼠动脉内皮细胞，并分为SVV干预组、阴性对照组、空白对照组。SVV干预组用腺病毒转染SVV-增强型绿色荧光蛋白（enhance green fluorescent protein，EGFP），阴性对照组转染空病毒，空白对照组不作处理。对细胞进行Transwell实验检测细胞迁移能力变化，酵素免疫分析法（enzyme-Linked ImmunoSorbent Assay，ELISA）检测基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）的含量变化，WST-1检测细胞增殖能力，流式细胞学检测细胞周期，Western blot分别检测SVV、EPAS1、CHOP-10、凋亡蛋白Caspase-8表达变化。用U0126和LY294002分别阻断EPAS1上游信号通路P44/42 丝裂原活化蛋白激酶（P44/42 mitogen-ac－tivated protein kinase，P44/42 MAPK）和磷酸肌醇3-激酶/AKT（phosphatidylinositide 3-kinase，PI3K/AKT）后，Western blot检测EPAS1表达，研究SVV影响EPAS1的机制。结果：过表达SVV能促进内皮细胞的细胞迁移能力，MMP-2、MMP-9分泌水平上升。转染SVV后细胞增殖能力增强，S期细胞增多，转染后SVV表达上升（F=326.137，P=0.000）。过表达组EPAS1蛋白相对表达量为1.29±0.11，阴性对照组为0.97±0.06，空白表达组为0.99±0.06，SVV能上调EPAS1表达，且有统计学差异（F=17.765，P=0.000）。过表达SVV能抑制CHOP-10表达，差异有统计学意义（F=78.908，P=0.000）。过表达SVV后Caspase-8相对表达量明显减少（F=53.823，P=0.000）。而阴性对照组和空白对照组的3种蛋白表达量无统计学差异（P=0.841，P=0.891，P=0.955）。通过阻断P13K/AKT途径能抑制SVV引起的EPAS1表达上调[（0.78±0.04） vs. （1.32±0.28），P=0.002]，P42/44 MAPK通路阻断剂U0126作用后EPAS1表达减少无统计学差异[（1.36±0.12） vs. （1.32±0.28），P=0.451]。结论：缺氧条件下SVV过表达促进细胞增殖、促进MMP-2、MMP-9分泌和提高细胞迁移能力。SVV主要通过P13K/AKT途径上调内皮细胞EPAS1表达，并抑制CHOP-10和Caspase-8表达，从而保护细胞适应低氧环境，发挥抗凋亡作用和潜在促血管生成的功能。

Abstract:

Objective：To investigate the effect of survivin（SVV） gene overexpression on the proliferation and migration of rat vascular endothelial cells with hypoxic treatment and the protein expression of EPAS1 and CHOP-10，as well as the possible mechanisms. Methods：Rat arterial endothelial cells were given hypoxic treatment with 500 μmol/L CoCl2 and were then divided into SVV inter－vention group，negative control group，and blank control group. The cells in the SVV intervention group were transfected with aden－ovirus SVV-EGFP，those in the negative control group were transfected with empty virus，and those in the blank control group were not given any treatment. Transwell assay was used to evaluate migration ability；ELISA was used to measure the changes in the levels of matrix metallopeptidase-2（MMP-2） and matrix metallopeptidase-9（MMP-9）；the WST-1 test was used to assess cell proliferation；flow cytometry was used to determine the cell cycle；Western blot was used to measure the changes in the protein expression of survivin，hypoxia-inducible factor EPAS1，endoplasmic reticulum stress protein CHOP-10，and apoptosis protein caspase-8. U0126 and LY294002 were used to block the EPAS1 upstream signaling pathways P44/42 MAPK and P13K/AKT，and then Western blot was used to measure the expression of EPAS1 to investigate the mechanism of the effect of survivin on EPAS1. Results：SVV overexpression promoted the migration ability of vascular endothelial cells and increased the secretion of MMP-2 and MMP-9. The proliferation ability of endothelial cells was enhanced after survivin trans－fection，with an increase in the number of cells in S phase，and there was an increase in the expression of survivin after transfection（F=326.137，P=0.000）. The relative protein expression of EPAS1 was 1.29±0.11 in the overexpression group，0.97±0.06 in the neg－ative control group，and 0.99±0.06 in the blank control group，suggesting that survivin significantly upregulated the expression of EPAS1（F=17.765，P=0.000）. SVV overexpression significantly inhibited the expression of CHOP-10（F=78.908，P=0.000）. There was a significant reduction in the relative expression of caspase-8 after survivin overexpression（F=53.823，P=0.000）. There were no significant differences in the expression of these three proteins between the negative control group and the blank control group（P=0.841，0.891，and 0.955）. The upregulated expression of EPAS1 due to survivin was inhibited by blocking the P13K/AKT pathway（0.78±0.04 vs. 1.32±0.28，P=0.002），and after U0126 was used to block the P42/44 MAPK pathway，there was a slight reduction in the expression of EPAS1（1.36±0.12 vs. 1.32±0.28，P=0.451）. Conclusion：Under the hypoxic condition，survivin overexpression can promote cell proliferation and migration abilities and increase the secretion of MMP-2 and MMP-9. SVVexerts an anti-apoptosis effect and promotes angiogenesis by upregulating the expression of EPAS1 in endothelial cells via the P13K/AKT pathway，inhibiting the expression of CHOP-10 and caspase-8，and protecting the cells against the hypoxic environment.