Introduction and Objective: Diabetes damages retinal mitochondria and impairs their removal by mitophagy, and these damaged mitochondria continue to accumulate. Diabetes also results in aberrant expression of many long noncoding RNAs (LncRNAs), the RNA transcripts with 200 nucleotides. These noncoding RNAs can interact with RNA, DNA and proteins, regulating many physiological processes. Among them, LncRNA HOTAIR, a highly conserved LncRNA implicated in oxidative stress, apoptosis and inflammation, is upregulated in the retina, vitreous and serum in diabetes. Our aim was to investigate the role of HOTAIR in mitophagy in diabetic retinopathy. Methods: Human retinal endothelial cells, untransfected or HOTAIR-siRNA transfected, incubated in 20mM D-glucose, were analyzed for mitophagy by quantifying mitophagy flux (flow cytometrically using Mtphagy Dye) and mitophagosome formation (LysoTraker staining for lysosome and MitoTracker green for mitochondria). Since mitophagy is closely associated with mtDNA biogenesis, mtDNA copy numbers (ratio of mtDNA-encoded CoxIV and nuclear DNA-encoded β-actin) were quantified. For mitochondrial function, mitochondrial membrane potential (JC-1 staining) and ROS levels (MitoSox red) were measured. Results: Compared to untransfected cells in normal glucose (5mM D-glucose), high glucose upregulated HOTAIR expression by ~75%. HOTAIR-siRNA, in addition to preventing glucose-induced increase in HOTAIR expression, ameliorated decrease in mitophagy flux and mitophagosome formation. In the same HOTAIR-siRNA transfected cells, glucose-induced decrease in mtDNA copy numbers, increase in mitochondrial ROS and impairments in mitochondrial potential were also improved. Conclusion: HOTAIR upregulation in diabetes impedes removal of the damaged mitochondria, and the dysfunctional mitochondria continue to produce damaging ROS. Thus, targeting HOTAIR to restore mitochondrial integrity could provide a fresh insight into the removal of the damaged mitochondria in diabetic retinopathy. Disclosure R. Kowluru: None. J. Kumar: None. P.B. Malaviya: None. Funding R01 014370R01 033516
KOWLURU et al. (Sun,) studied this question.