RESUMO
Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Fator de Transcrição E2F1/antagonistas & inibidores , Neovascularização Fisiológica , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/fisiologia , Células Endoteliais/metabolismo , Efrina-B2/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Cultura Primária de Células , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Artérias Umbilicais/metabolismoRESUMO
The corneal thickness of the center and other 24 points were measured in 31 normal eyes and 246 myopic eyes. The results show that the mean central corneal thickness of the normal eye is 541 +/- 45.29 microns and that of the myopic eye, 544 +/- 35.01 microns. There are no significant differences in the central corneal thickness in comparisons between the left and right eye, between the male and female, among various age groups, and among various degrees of refractive error. However, with the increase of age and increase of myopic refractive error, the central corneal thickness gradually becomes thinner. There are differences in thickness among various directions, the superior direction being the thickest and the temporal inferior, the thinnest. From the center to the limbus, the thickness of the cornea gradually increases, but the values of the increase of the thickness are various in various directions, that should be paid attention to in radial keratotomy.
