Abstract
Non-POU domain containing octamer binding (NONO) protein is a key player in the paraspeckle complex and is engaged in a number of biological processes. Additionally, nothing is known about the transcriptional controls of the NONO gene. This study investigates the regulation of NONO gene expression in HUVEC (human umbilical vein endothelial cells) under hypoxic and normoxic conditions and the influence of VEGF cytokine. The chemically simulated hypoxic state was confirmed by measuring a 5-fold increase in HIF1-α mRNA levels compared to normoxic conditions. Additionally, NONO mRNA levels were significantly upregulated after 6 hours of hypoxia treatment. To determine the basal promoter activity of the NONO gene, different promoter fragments (P1: -730/+529, P2: -516/+529, P3: -336/+529, and P4: -159/+529) were transiently transfected into HUVEC cells under both normoxic and hypoxic conditions. The results showed that all promoter constructs were transcriptionally active, with the largest promoter construct (P1: -730/+529) exhibiting the highest activity in both conditions. This suggests that the − 730/+529 promoter region is crucial for the increased gene expression observed in hypoxic conditions. qRT-PCR results demonstrated that VEGF significantly increased NONO mRNA levels, with the highest expression observed at 24 hours. Specifically, VEGF treatment resulted in a 2-fold increase in NONO mRNA levels under normoxic conditions at 24 and 48 hours and a 5-fold increase under hypoxic conditions at 24 hours. At the protein level, VEGF induced a 2-fold increase at 24 hours under normoxia and a 3-fold increase at 48 hours under hypoxia. Pathway inhibition studies identified the specific pathways involved in VEGF-induced NONO expression. Inhibitors targeting JNK, PI3K, and ERK1/ERK2 pathways significantly reduced VEGF-mediated NONO expression at both mRNA and protein levels. This indicates that these pathways play a critical role in the upregulation of NONO gene expression by VEGF.Overall, the study provides essential insights into the molecular mechanisms by which hypoxia and VEGF regulate NONO gene expression in endothelial cells, highlighting the potential therapeutic targets for diseases involving endothelial cell dysfunction and angiogenesis.