角质细胞叉头框蛋白O1基因调节血管内皮生长因子A的表达

Forkhead box O1 gene in keratinocytes regulates expression of vascular endothelial growth factor-A

目的通过体外实验在角质细胞中过表达或沉默叉头框蛋白O1(FOXO1)基因,研究其对血管内皮生长因子A(VEGF-A)蛋白水平和转录活性的影响。方法采用体外细胞培养和转染方法,分别用ON-TARGET plus SMART pool人FOXO1siRNA和对照siRNA(ON-TARGET plus Non-targeting Control Pool)转染人永生化牙龈角质细胞(human immortalized gingival keratinocyte,HIGK),用免疫荧光法检测HIGK中FOXO1和VEGF-A的免疫荧光强度,荧光酶素实验分别检测FOXO1过表达和FOXO1沉默时VEGF-A的活性。结果与未转染组相比,使用FOXO1siRNA转染的HIGK中VEGF-A的蛋白水平降低了57%(P<0.05)。荧光酶素实验分析结果显示,在HIGK中过表达FOXO1后VEGF-A的转录活性增加了2.1倍(P<0.05),而FOXO1沉默时VEGF-A的转录活性减少了20%~43%(P<0.05)。结论角质细胞中FOXO1参与VEGF-A的表达调控。

Objective To explore the effect of forkhead box O1（FOXO1）gene on the expression and transcription of vascular endothelial growth factor-A（VEGF-A）through overexpressing or silencing the FOXO1 gene in vitro.Methods The human immortalized gingival keratinocyte（HIGK）was cultured and transfected with ON-TARGET plus SMART pool for human FOXO1 siRNA or ON-TARGET plus Non-targeting Control Pool（control siRNA）,the fluorescence intensity of VEGF-A was measured by the immunofluorescence staining in vitro.Luciferase assay was used to test the VEGF-A luciferase activity by FOXO1 overexpression or FOXO1 silence.Results The VEGF-A protein levels in HIGK transfected with FOXO1 siRNA significantly reduced by 57%compared with HIGK without transfection（P〈0.05）.The luciferase reporter results showed that overexpression of FOXO1 resulted in a 2.1-fold increase in VEGF-A transcriptional activity（P〈0.05）,and FOXO1 silencing produced a 20%-43% decrease in VEGF-A transcriptional activity（P〈0.05）.Conclusion FOXO1 in HIGKs can regulate VEGF-A protein expression.