Abstract
Purpose :
Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein that is upregulated in endothelial and epithelial cells during inflammation. ICAM-1 promotes leukocyte transendothelial migration and plays a role in angiogenesis. The lncRNA ICR (CTD-2369P2.8) is correlated with ICAM-1 mRNA stabilization in hepatocellular carcinoma. MALAT1 and MEG3 lncRNAs are associated with microvascular complications and ICAM-1 induced inflammation in diabetic retinopathy. We investigated lncRNA expression in human retinal endothelial cells (hREC) and retinal pigment epithelial (ARPE-19) cells and analyzed the ICR promoter for response to TNFα and LPS.
Methods :
Primary hREC were isolated from 4 cadaveric donors and, along with ARPE-19 cells, were stimulated with either TNFα (10 ng/ml) or LPS (10 μg/ml) for 4 hrs. Total RNA was isolated and cDNA prepared. Quantitative RT-PCR was used to assess changes in gene expression. Genomatix gene regulation software was used to analyse the ICR promoter region. ICR promoter fragments of 669 bp and 1069 bp were cloned into pNLuc plasmids for reporter assays using the Nan-Glo Luciferase system. Transfection efficiency was optimized by varying concentrations of and incubation times with Lipofectamine 2000.
Results :
ICR was induced in hREC by TNFα, but MALAT1 and MEG3 were not. Increase in ICR in response to TNFα varied between donors, ranging from 10-, 22-, 33- and 73-fold (P<0.01, n=4). There was a positive correlation between levels of ICR transcript and ICAM-1 expression. LPS did not induce ICR in REC but did increase ICR expression (5-fold) in ARPE-19 cells (P<0.01, n=3). Analysis of the human ICR promoter identified a 117bp evolutionary conserved region with 23 other mammalian species, situated within 600 bp of the TSS. The 669 bp ICR promoter fragment was not responsive to TNFα or LPS. The 1069 bp promoter region was responsive to TNFα in hREC (11-fold increase, n=8, P<0.05).
Conclusions :
Our results indicate that ICR is expressed in hREC and is induced by an inflammatory cytokine. Future experiments will determine the regulatory elements within the ICR promoter that respond to TNFα and whether ICR influences ICAM-1 expression. Understanding the regulation of lncRNA expression in ocular cells during ICAM-1 associated pathology may open new avenues of therapy for inflammatory or angiogenic diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.