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

目的: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500μmol/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-activated 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。

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 EPAS1and CHOP-10,as well as the possible mechanisms. Methods:Rat arterial endothelial cells were given hypoxic treatment with 500txmol/L COC12and were then divided into SVV intervention group,negative control group,and blank control group.The cells in the SVV intervention group were transfected with adenovirus 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 EPAS 1,endoplasmic reticulum stress protein CHOP-10,and apoptosis protein caspase-8.U0126and LY294002were used to block the EPAS1 upstream signaling pathways P44/42MAPK 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 transfection,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±0.11in the overexpression group,0.97±0.06in the neg- ative control group,and 0.99±0.06in the blank control group,suggesting that survivin significantly upregnlated 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-8after 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 EPAS1due to survivin was inhibited by blocking the P13K/AKT pathway (0.78±0.04vs.1.32±0.28,P=0.002),and after U0126was used to block the P42/44MAPK pathway,there was a slight reduction in the expression of EPAS1(1.36±0.12vs.1.32±0.28,P=0.451). Conclusion :Under the hypoxic condition,survivin overexpression dan promote cell proliferation and migration abilities and increase the secretion of MMP-2and 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.