Abstract
Purpose :
Mitochondrial DNA (mtDNA), a 16.7kb circular DNA, is damaged in diabetic retinopathy and its transcription is impaired, and the electron transport chain is compromised, fueling into the vicious cycle of free radicals. Diabetes also alters expressions of many long non-coding RNAs (LncRNAs), the RNAs with more than 200 nucleotides and no open reading frame for translation. Although most of the LncRNAs are encoded by nuclear genome, mitochondrial genome also encodes three LncRNAs, and the expression of mtDNA-encoded LncRNA Cytochrome B (LnCytB) is decreased in diabetes. The aim of this study was to investigate the role of LncCytB in mtDNA stability in diabetic retinopathy.
Methods :
Human retinal endothelial cells, incubated in normal (5mM) or high (20mM) D-glucose for four days, were analyzed for LncCytB expression by qRT-PCR and strand-specific PCR, and its mitochondrial localization by RNA fluorescence in situ hybridization (RNA-FISH)-immunofluorescence technique. Packaging of the mtDNA was determined by Flow Cytometry and by its sensitivity to Micrococcal nuclease digestion. The role of LncCytB in mtDNA stability was confirmed in HRECs transfected with LncCytB by quantifying mtDNA damage (by extended length PCR) and its protective nucleoids (by SYBR green staining).
Results :
Compared to normal glucose, high glucose decreased LnCytB levels in the mitochondria. While the sensitivity of mtDNA to Micrococcal nuclease was increased, packaging of mtDNA and its protective nucleoids were decreased. Overexpression of LnCytB ameliorated glucose-induced increase in mtDNA damage and decrease in mtDNA-encoded nucleoids.
Conclusions :
Downregulation of LncCytB in the mitochondria in hyperglycemic milieu reduces the protective nucleoids, which makes mtDNA more susceptible to the damage. The damaged mtDNA compromises the electron transport chain and the vicious cycle of free radicals continues to self-propagate. Thus, regulation of LnCytB has potential to prevent mtDNA damage, and the progression of diabetic retinopathy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.