Abstract
Purpose :
Mitochondria play an important role in the development of diabetic retinopathy, and their dysfunction initiates capillary cell apoptosis. Diabetes also alters the levels of many long non-coding RNAs (LncRNAs, the RNAs with >200 nucleotides and no open reading frame for translation). Although majority of the LncRNAs are encoded by nuclear DNA, mtDNA also encodes three LncRNAs, and our recent study has shown that mtDNA-encoded LncRNA Cytochrome B (LncCytB) is downregulated in diabetes, impairing mitochondrial genomic stability. Mitochondrial-targeted RNase P protein 1, MRPP1, cleaves and processes mitochondrial transcripts, and is also implicated in the regulation of mtDNA-encoded LncRNAs. Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy.
Methods :
MRPP1 and LncCytB expressions were quantified in the human retinal endothelial cells (HRECs), incubated in normal (5mM; NG) or high (20mM; HG) D-glucose for 96 hours by qRT-PCR and strand-specific PCR, and their mitochondrial localization by western blotting and RNA fluorescence in situ hybridization (RNA-FISH), respectively. The interactions between MRPP1 and LncCytB were measured by RNA-FISH and confirmed by RNA immunoprecipitation assay. The occupancy of LncCytB at MRPP1 promoter was quantified by chromatin isolation by RNA purification technique. In vitro results were confirmed in the retinal microvessels from human donors with documented diabetic retinopathy.
Results :
Compared to normal glucose, cells in high glucose had reduced MRPP1 and LncCytB expressions, and their mitochondrial localization was decreased. Interactions between MRPP1 and LncCytB were reduced, and LncCytB occupancy at MRPP1 promoter was also decreased. Consistent with HRECs, retinal microvessels from donors with diabetic retinopathy had reduced levels of MRPP1 and LncCytB, and decreased interactions between them.
Conclusions :
Thus, due to impaired interaction of LncCytB with MRPP1 in diabetes, LncCytB levels are decreased in the mitochondria, and this compromises mitochondrial function, leading to the development of diabetic retinopathy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.