Abstract
Diabetic retinopathy, a microvascular complication of diabetes, is the leading cause of blindness in adults, but the molecular mechanism of its development remains unclear. Retinal mitochondrial DNA is damaged and hypermethylated, and mtDNA-encoded genes are downregulated. Expression of a long noncoding RNA (larger than 200 nucleotides, which does not translate into proteins), encoded by mtDNA, cytochrome B (LncCytB), is also downregulated. This study aims to investigate the role of DNA methylation in the downregulation of LncCytB in diabetic retinopathy. Human retinal endothelial cells, incubated in 5 mM (normal) or 20 mM (high) D-glucose, in the presence/absence of Azacytidine (a DNA methyl transferase inhibitor) were analyzed for LncCytB DNA methylation by immunoprecipitation and methylation specific PCR techniques, and LncCytB transcripts by strand-specific PCR and RNA-FISH. Mitochondrial genomic stability was evaluated by quantifying protective mtDNA nucleoids by SYBR green staining and by flow cytometry, and functional stability by oxygen consumption rate using Seahorse analyzer. Results were confirmed in an in vivo model using retina from diabetic rat. While high glucose elevated 5 mC and the ratio of methylated to unmethylated amplicons at LncCytB and downregulated its transcripts, azacytidine prevented LncCytB DNA hypermethylation and decrease in its expression. Azacytidine also ameliorated decrease in nucleoids and oxygen consumption rate. Similarly, azacytidine prevented increase in retinal LncCytB DNA methylation and decrease in its expression in diabetic rats. Thus, DNA hypermethylation plays a major role in the downregulation of retinal LncCytB in diabetes, resulting in impaired mitochondrial homeostasis, and culminating in the development of diabetic retinopathy.