Abstract
Purpose:
To observe the nuclear expression of heparanase(Hpa, an endoglucuronidase that specifi-cally cleaves carbohydrate chains of heparan sulfate) and RNA polymerase II (Pol II, an enzyme that catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA) in human retinal microvascular endothelial cells (HRECs) under hyperglucose condition and to investigate the association of heparanase with the transcription activity of VEGF gene promoter.
Methods:
Cultured HRECs were maintained for 3 days in media with 30mmol/L glucose or normal glucose.The expression of heparanase, Pol II in each group was analyzed with double immunofluorescence. Cells in both groups were used for Chromatin Immunoprecipitation (ChIP) with anti-Hpa and anti-Pol II antibodies to identify high-confidence Hpa-binding regions across the entire VEGF gene promoter, and use real-time PCR to demonstrate the interaction between heparanase and the VEGF gene promoter region.
Results:
Double immunofluorescence studies showed that the expression of heparanase in cytoplasm and nuclear was intense in hyperglucose HRECs, but faint in normal group; heparanase and Pol II in nucleu was also intense in hyperglucose HRECs, and the distribution of heparanase is consistent with that of Pol II. Real-time PCR-based ChIP results showed the high-confidence Hpa-binding regions was -1155 to -1018 (containing hypoxia response element) in VEGF gene promoter, and the cells treated with 30 mmol/L glucose showed an increase of heparanase and Pol II in VEGF gene promoter region, compared with the normal glucose treated cells (t=-3.244, P=0.032; t=-6.096, P=0.004, respectively).
Conclusions:
Nuclear heparanase is directly combined to VEGF gene promoter, and involved in the regulation of VEGF gene transcription in hyperglucose HRECs.
Keywords: 748 vascular endothelial growth factor •
738 transcription •
533 gene/expression