ZNF580在1-磷酸鞘氨醇诱导内皮细胞迁移和增殖中的作用

ZNF580 is up-regulated in S1P-induced migration and proliferation of endothelial cells

目的:研究人锌指蛋白ZNF580在1-磷酸鞘氨醇(sphingosine 1-phosphate,S1P)诱导内皮细胞迁移和增殖中的作用,为探讨ZNF580功能提供科学依据。方法:RT-PCR检测S1P受体在EA.hy926细胞的表达情况;不同浓度(0～10μmol/L)S1P刺激EA.hy926细胞不同时间(0～12 h)后,RT-PCR和Western blotting检测S1P对ZNF580表达的影响;利用p38 MAPK信号通路特异性抑制剂SB203580研究S1P是否通过此信号通路影响ZNF580的表达;脂质体转染法获得瞬时过表达和瞬时低表达ZNF580的EA.hy926细胞;Transwell实验及MTT比色法分析ZNF580对内皮细胞迁移和增殖活性的影响。结果:EA.hy926细胞表达S1P1、S1P3和S1P5三种受体,其cDNA的特异性扩增产物分别为352 bp、701 bp和236 bp;S1P刺激EA.hy926细胞后,ZNF580的表达呈现剂量和时间依赖性增高;SB203580能够抑制S1P诱导的ZNF580的上调作用;ZNF580过表达(低表达)后内皮细胞迁移和增殖活性明显增强(减弱)。结论:S1P通过p38 MAPK信号通路影响ZNF580的表达;ZNF580在内皮细胞迁移和增殖过程中起着重要的调控作用。

AIM： To investigate whether a novel human C2H2 --type zinc finger protein ZNF580 is involved in the proliferation and migration of endothelial cells induced by sphingosine 1 -phosphate （SIP）. METHODS： The eDNA of EA. hy926 cells was analyzed by RT - PCR to determine the S1P receptor expression profile. The cells were incubated with SIP at different concentrations and for different time intervals. Total RNA and protein in treated EA. hy926 cells were analyzed by RT - PCR and Western blotting. SB203580, a chemical inhibitor of p38 MAPK, was used to determine wheth- er p38 MAPK pathway had any effect on the up -regulation of ZNF580 expression by SIP. The plasmid pEGFP -ZNF580 or the synthetic ZNF580 - siRNA was transfected into EA. hy926 cells with Lipofectamine 2000 for 48 h. Cell migration as- say and MTT colorimetrie assay were used to investigate the effects of ZNF580 on the motility and growth of endothelial cells. RESULTS： EA. hy926 endothelial cells expressed S1P1, S1 P3 and S1 P5 receptors. Furthermore, S1P up - regula- ted ZNF580 at mRNA and protein levels in a concentration - and time - dependent manner. The p38 MAPK pathway spe- cific inhibitor SB203580 blocked the S1P - induced up - regulation of ZNF580 expression. Moreover, overexpression/silen- cing of ZNF580 in EA. hy926 cells led to enhancement/decrease of the migration and proliferation of the cells. CONCLU- SION： SIP - induced migration and proliferation of endothelial cells are critical for angiogenesis. ZNF proteins usually play an essential role in altering gene expression and regulating the angiogenesis.