乙酰肝素酶介导高糖环境下人视网膜微血管内皮细胞中血管内皮生长因子基因的转录。

Heparanase mediates vascular endothelial growth factor gene transcription in high-glucose human retinal microvascular endothelial cells.

作者信息

Hu Jie, Wang Jingwei, Leng Xuan, Hu Yijun, Shen Huangxuan, Song Xin

机构信息

The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

出版信息

Mol Vis. 2017 Aug 10;23:579-587. eCollection 2017.

PMID:28848320

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5561135/

Abstract

PURPOSE

To observe the nuclear expression and interaction of heparanase and RNA polymerase II (RNA Pol II), an enzyme that catalyzes the transcription of DNA in eukaryotic cells) in human retinal microvascular endothelial cells (HRECs) under high glucose condition and to investigate the association of heparanase with the transcription activity of the vascular endothelial growth factor () gene promoter.

METHODS

Cultured HRECs were maintained for 3 days in media with high or normal glucose. The expressions of heparanase and RNA Pol II in each group were analyzed with immunofluorescence. Co-immunoprecipitation was applied to detect the interaction of heparanase and Pol II proteins. Cells in both groups were used for chromatin immunoprecipitation (ChIP) with anti-heparanase and anti-RNA Pol II antibodies to identify high-confidence heparanase-binding regions across the entire gene promoter. Moreover, real-time PCR was used to demonstrate the interaction between heparanase and the gene promoter region.

RESULTS

The immunofluorescence studies showed that the nuclear expression of heparanase was intense in high-glucose HRECs but faint in the normal group; RNA Pol II in the nucleus was also intense in high glucose HRECs, and the distribution of heparanase was consistent with that of RNA Pol II. The co-immunoprecipitation data showed that heparanase combined with RNA Pol II in HRECs cells treated with high glucose, and the molecular size of HPA interacted with RNA Pol II was 50 kDa, while no combination of two proteins was evident in normal HRECs cells. Real-time PCR-based ChIP results showed that the high-confidence HPA-binding region was -1155 to -1018 (containing hypoxia response element) in the VEGF gene promoter, and the cells treated with high glucose showed increases in heparanase and RNA Pol II in the gene promoter region compared with the normal glucose treated cells ( = -3.244, = 0.032; = -6.096, = 0.004, respectively).

CONCLUSIONS

Nuclear heparanase combines directly with the gene promoter and is involved in the regulation of gene transcription in high-glucose HRECs.

摘要

目的

观察高糖条件下人视网膜微血管内皮细胞（HRECs）中乙酰肝素酶与RNA聚合酶II（RNA Pol II，一种在真核细胞中催化DNA转录的酶）的核表达及相互作用，并探讨乙酰肝素酶与血管内皮生长因子（VEGF）基因启动子转录活性的关系。

方法

将培养的HRECs在高糖或正常葡萄糖培养基中培养3天。用免疫荧光法分析每组中乙酰肝素酶和RNA Pol II的表达。采用免疫共沉淀法检测乙酰肝素酶与Pol II蛋白的相互作用。两组细胞均用抗乙酰肝素酶和抗RNA Pol II抗体进行染色质免疫沉淀（ChIP），以鉴定整个VEGF基因启动子上的高可信度乙酰肝素酶结合区域。此外，用实时PCR证明乙酰肝素酶与VEGF基因启动子区域之间的相互作用。

结果

免疫荧光研究显示，高糖HRECs中乙酰肝素酶的核表达强烈，而正常组中则较弱；高糖HRECs中细胞核内的RNA Pol II也强烈，且乙酰肝素酶的分布与RNA Pol II一致。免疫共沉淀数据显示，高糖处理的HRECs细胞中乙酰肝素酶与RNA Pol II结合，与RNA Pol II相互作用的HPA分子大小为50 kDa，而正常HRECs细胞中未发现两种蛋白结合。基于实时PCR的ChIP结果显示，VEGF基因启动子中高可信度的HPA结合区域为-1155至-1018（含缺氧反应元件），与正常葡萄糖处理的细胞相比，高糖处理的细胞在VEGF基因启动子区域的乙酰肝素酶和RNA Pol II增加（分别为t = -3.244，P = 0.032；t = -6.096，P = 0.004）。