Background Anti-VEGF drugs are generally applied in the treatment of ocular neovascular diseases.However,the therapy effect is unsatisfactory in some patients.Studing the effect of hypoxia-inducible factor-1 (HIF-1),a upstream regulatory gene of VEGF,and its limiting enzyme prolyl-4-hydroxylase domain proteins (PHDs) is of important clinical significance.Objective This study was to investigate the negtive regulation of exogeneous PHDs on HIF-1 pathway in human RPE cells.Methods pFLAG-PHD1,pFLAG-PHD2 and pFLAG-PHD3 plasmids were constructed by extracting RNA from Hela cell line and coloning PHD1,PHD2 and PHD3 using reverse transcription PCR with restriction enzyme.The plasmids were identified by gene sequencing.ARPE-19 cells were cultured at 21％ O2 (normoxia group),1％ O2 (hypoxia group),or in hypoxia-mimicking agents (CoCl2,anoxia group),respectively,and then were transfected with plasmids encoding FLAG-tagged PHD1,PHD2,PHD3 and pFLAGCMV2 transfected cells served as blank control.The expressional intensities of PHD1,PHD2 and PHD3 in the cells were detected and compared among different groups by using Western blot assay.The transcriptional activity of HIF-1 in the cells was evaluated with dual luciierase reporter assay.Results Western blot assay showed that PHD1,PHD2 and PHD3 all were expressed in ARPE-19 cells in the normoxia group,hypoxia group and anoxia group.The expression was strong in PHD2 protein and was weak in PHD3 protein,a statistically significant difference was found between PHD2 protein expression and PHD1 or PHD3 expressions (all at P＜0.05).Endogenous HIF-1 activity was elevated in pFLAG-CMX transfected cells in the hypoxia group and anoxia group than that in the normoxia group.Compared with pFLAG-CMX transfected cells,no obvious change was seen in the endogenous HIF-1 activity in the normoxia group,however,HIF-1 activity was declined in the hypoxia group and anoxia group after pFLAG-PHD1,pFLAG-PHD2 or pFLAG-PHD3 transfection.Under the same oxygen environment,HIF-1 activity was lower in the pFLAG-PHD2 transfected cells than that in the pFLAG-PHD1 or pFLAG-PHD3 transfected cells (both at P＜0.05).Conclusions PHDs play a negative regulation to HIF-1 activating pathway in human RPE cells,especially in hypoxia and anoxia cells.Among PHDs proteins,PHD2 presents the strongest inhibition on HIF-1 activating pathway.%背景 目前抗VEGF药物的应用已广泛用于眼部新生血管性疾病发病的治疗,但有部分患者的疗效并不理想,因此研究VEGF的上游基因缺氧诱导因子-1(HIF-1)及其限速酶脯氨酸羟化酶(PHDs)在新生血管形成中的作用具有重要的临床意义. 目的 探讨外源性PHDs在HIF-1激活通路中的负性调节作用.方法用Hela细胞提取RNA,采用逆转录PCR法从cDNA克隆PHD1、PHD2和PHD3,通过限制性内切酶构建pFLAG-PHD1、pFLAG-PHD2和pFLAG-PHD3质粒并通过基因测序进行鉴定.分别将人RPE细胞株(ARPE-19)在体积分数21％ O2(常氧组)、1％ O2(低氧组)和缺氧模拟剂(CoCl2,缺氧组)条件下进行培养,将pFLAG-PHD1、pFLAG-PHD2和pFLAG-PHD3质粒分别转染至培养的ARPE-19细胞中,pFLAG-CMV2转染作为空白对照.采用Western blot法测定和比较转染细胞在不同氧环境培养下PHD1、PHD2和PHD3蛋白的表达强度;采用双荧光素酶报告系统评估各组细胞中HIF-1的转录活性. 结果 Western blot法检测显示常氧组、低氧组和缺氧组ARPE-19细胞中均有PHD1、PHD2和PHD3蛋白的表达,各组细胞中PHD2的表达条带均强于PHD1和PHD3蛋白,差异均有统计学意义(P＜0.05).pFLAG-CMX转染后常氧组细胞中内源性HIF-1活性反应低,低氧组和缺氧组细胞中HIF-1的转录活性明显升高,与空白对照组相比,pFLAG-PHD1、pFLAG-PHD2、pFLAG-PHD3转染后常氧组细胞中内源性HIF-1活性反应无明显变化(F=0.48,P＞0.05),而低氧及缺氧组细胞中HIF-1活性明显下降,与空白对照组比较差异均有统计学意义(F=24.30,112.67,均P＜0.05).相同培养条件下,pFLAG-PHD2转染后细胞中HIF-1活性明显低于pFLAG-PHD1和pFLAG-PHD3转染细胞,差异均有统计学意义(均P＜0.05). 结论 PHDs对人RPE细胞中HIF-1的激活通路有明显的负向调节作用,其对低氧细胞和缺氧细胞中HIF-1转录活性的抑制作用明显强于常氧细胞,其中PHD2抑制HIF调节通路的作用明显强于PHD1和PHD3.
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