Abstract
Purpose :
Diabetic retinopathy is a sight threatening outcome of prolonged hyperglycaemia and oxidative stress is one of the major factor for its pathogenesis and progression. Nuclear factor erythroid 2-related factor, NRF2, plays a major role in regulating transcription of many antioxidant defence genes. In diabetes, due to increased levels of its intracellular inhibitor, Kelch-like ECH-associated protein 1 (Keap1), NRF2 nuclear translocation is decreased. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) lncRNA is shown to contribute in NRF2 and Keap1 pathway by gene regulation. Our aim is to identify the link between MALAT1 activation and failure of antioxidant defence system in diabetic retinopathy.
Methods :
Human retinal endothelial cells (HRECs) were incubated in high glucose (20 mM) for 96 hours. Epigenetic regulation of Keap1 was investigated by quantifying the binding of the transcription factor SP1 at its promoter using Chromatin Immunoprecipitation (ChIP) technique. Role of MALAT1 in Keap1 activation was determined by quantifying the co-occupancy of both MALAT1 and SP1 on Keap 1 promoter by Chromatin Isolation by RNA Purification (ChIRP)- reChIP technique. Effect of MALAT1-siRNA on Keap1-NRF2 interactions and NRF2 nuclear localization was monitored by immunofluorescence. In the same siRNA transfected cells, MALAT1-Keap1-mediated transcriptional activation was quantified by qRT-PCR of NRF2 responsive genes superoxide dismutase 2 (Sod2) and heme oxygenase 1 (HO1).
Results :
Compared to normal glucose (5 mM), HRECs in high glucose had increased MALAT1 and Keap1 expressions, and Keap1 promoter had increased co-occupancy of SP1 and MALAT1. Regulation of MALAT1 by its siRNA ameliorated glucose-induced increase in Keap1 and decreased SP1 occupancy at its promoter. MALAT-siRNA also attenuated glucose-induced Keap1-NRF2 co-localization in the cytosol, and restored NRF2 translocation inside the nucleus and the expression Sod2 and HO1.
Conclusions :
MALAT1 plays a crucial role in maintaining the integrity of antioxidant defence mechanism via regulating Keap1. Thus, downregulation of MALAT1 has potential to restore NRF2 and ameliorating the development of retinopathy in diabetic patients.
This is a 2020 ARVO Annual Meeting abstract.