Retinal oxidative stress is increased and the antioxidant defence system is compromised in diabetic retinopathy. Expression of many key antioxidant genes is regulated by a master regulator, nuclear factor erythroid 2-related factor (Nrf2). The transcriptional activity of Nrf2 is mediated by an intracellular inhibitor Kelch-like ECH-associated protein 1 (Keap1), which regulates its nuclear movement. In diabetes, Nrf2 transcriptional activity is decreased and Keap1 levels are increased. A long noncoding RNA (IncRNA), Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), plays a role in regulating Keap1 expression, and retinal MALAT1 levels are upregulated in diabetes. Our aim is to investigate the role of MALAT1 in maintaining the antioxidant defence system in diabetic retinopathy. Human retinal endothelial cells (untransfected or transfected with MALAT1-siRNA) were incubated in 20mM glucose for 96 hours. MALAT1 expression was analysed by q-RTPCR and RNA sequencing, and role of MALAT1 in Keap1 transcription was quantified using MALAT1 RNA pull down assay. Simultaneous occupancy of MALAT1 and transcription factor Sp1 on Keap1 promoter was quantified by Chromatin Isolation by RNA Purification (ChIRP)-reChIP technique, nuclear translocation of Nrf2 by immunofluorescence imaging, and Nrf2-responsive genes superoxide dismutase 2 (Sod2) and heme oxygenase 1 (HO1) by q-RTPCR. Compared to cells in 5mM glucose, high glucose increased co-occupancy of MALAT1 and Sp1 at Keap1 promoter. MALAT1-siRNA ameliorated glucose-induced increase in co-occupancy of MALAT1-Sp1 at itspromoter and decreased Keap1 expression. MALAT1-siRNA also restored nuclear localization of Nrf2 and the transcription of Sod2 and HO1. Thus, IncRNA MALAT1, via Keap1, modulates the transcriptional activity of Nrf2 and the antioxidant defence system in diabetic retinopathy. Regulation of MALAT1 can protect the retina from oxidative damage, and prevent/slow down the development of diabetic retinopathy.
R. Kowluru: None. R. Radhakrishnan: None.
EY014370, EY017313, EY022230