MP35-03 TWIST UP-REGULATES VEGF EXPRESSION WITH HIF IN HYPOXIA

Abstract

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2014MP35-03 TWIST UP-REGULATES VEGF EXPRESSION WITH HIF IN HYPOXIA Takeshi Yoshida, Ryoichi Hamasuna, Naohiro Fujimoto, and Tetsuro Matsumoto Takeshi YoshidaTakeshi Yoshida More articles by this author , Ryoichi HamasunaRyoichi Hamasuna More articles by this author , Naohiro FujimotoNaohiro Fujimoto More articles by this author , and Tetsuro MatsumotoTetsuro Matsumoto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1046AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES VHL (von-hippel-lindaw protein), which is E3 ubiquitin protein ligase, interacts with HIF1a (hypoxia-inducible factor-1a) and degrades HIF leading to suppress transcriptional activation of HIF target gene in normoxia condition. In hypoxia, HIF is not degraded by VHL and activate downstream genes such as VEGF. Nowadays several kinds of molecular target agents against VEGF signaling pathway are developed. But molecular target agents are limited in its effect. There are much more unknown molecules controlling this pathway. In this study, we focused on Twist, which is involved in EMT (epithelial-mesenchymal transition) and demonstrated its effect on VHL-HIF signaling pathway. METHODS Twist, siRNA Twist and other proteins was investigated in the human renal tumor cell line ACHN. Transcriptional activity of VEGF by Twist was measured by reporter assay using HIF responsive element (HRE) reporter plasmids co-transfected with Twist expression plasmid (or siRNA-Twist) in renal tumor cell line ACHN and other cell lines. Binding of Twist to VEGF promoter in tumor cells were shown by chromatin immunoprecipitation (ChIP) assay using antibody against Twist. Immunoprecipitated cleaved-DNA was amplified by PCR using primers coded with VEGF promoter sequence. Electrophoresis Mobility Shift Assay (EMSA) were demonstrated to show binding of Twist for specific sequence (about -1000bp upstream from start point of transcription) in VEGF promoter. To show molecular binding between Twist and HIF, Co-immunoprecipitation (Co-IP) assay were performed. Expression of VEGF by Twist was investigated by western assay and immuno-fluorescent staining in tumor cell line. RESULTS Overexpressed Twist activated HRE reporter activity. VEGF promoter activity was activated by Twist in hypoxia. ChIP assay showed Twist binding to VEGF promoter in ACHN cells. Co-IP assay showed the binding of Twist and HIF. VEGF was overexpressed in Twist transfected cells. CONCLUSIONS We report the association of Twist with VHL-HIF signaling pathway. This suggests that Twist expression involves in tumor vascularization in renal tumor. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e371 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Takeshi Yoshida More articles by this author Ryoichi Hamasuna More articles by this author Naohiro Fujimoto More articles by this author Tetsuro Matsumoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...