Mitochondrial genome-encoded long noncoding RNA and mitochondrial stability in diabetic retinopathy

<p>Mitochondria experience genomic and functional instability in diabetes, and mitochondrial dysfunction has a critical role in the development of diabetic retinopathy. Diabetes also alters expressions of many long non-coding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame. LncRNAs are mainly encoded by nuclear genome, but mtDNA also encodes three LncRNAs. Our goal was to investigate the effect of hyperglycemia on mtDNA-encoded LncRNA <em>Cytochrome B</em> (Lnc<em>CytB</em>) in mtDNA stability in diabetic retinopathy. Retinal endothelial cells, transfected with Lnc<em>CytB</em>- overexpressing plasmids or siRNA, incubated in 5mmols/L (NG) or 20mmols/L (HG) D-glucose for four days, were analyzed for Lnc<em>CytB </em>expression by strand-specific PCR, and its mitochondrial localization by RNA-FISH. Damage sensitive mtDNA regions were examined by micrococcal nuclease digestion-sequencing, and Lnc<em>CytB</em> occupancy at mtDNA by chromatin isolation by RNA purification. Protective nucleoids in mtDNA were analyzed by SYBR green-MitoTracker Red staining, and confirmed in isolated mitochondria by flow cytometry. Compared to NG, HG downregulated Lnc<em>CytB</em> by >50%, but had no significant effect on the other mtDNA-encoded LncRNAs. mtDNA packaging was impaired and MNase sensitivity was increased, and Lnc<em>CytB</em> occupancy at mtDNA was decreased. While Lnc<em>CytB</em> overexpression ameliorated mtDNA damage and decrease in nucleoids and copy numbers, Lnc<em>CytB</em>-siRNA exacerbated damage and further reduced nucleoids. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy presented similar decrease in Lnc<em>CytB</em> and mtDNA nucleoids. Thus, Lnc<em>CytB</em> has a major role in maintaining mitochondrial genomic stability, and its downregulation in hyperglycemic milieu contributes to increased vulnerability of mtDNA to damage.</p>