DNA methylation of long noncoding RNA cytochrome B in diabetic retinopathy

Figures

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  Fig. 1. Promoter region of LncCytB. (a) CpG island between 903 and 1085 bp (start to end, in red), and (b) amplification plot of the region used for DNA methylation of LncCytB.

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  Fig. 2. Effect of high glucose on DNA methylation of LncCytB: Cells incubated in high glucose for (a) 12–96 h were analyzed for 5 mC levels using the primers targeting CpG region of LncCytB in 5 mC antibody immunoprecipitated genomic DNA, and (b) for 96 h were used to determine the ratio of methylated and unmethylated amplicons in the bisulfite converted DNA. Values are represented as mean ± SD obtained from three independent cell preparations, and each assay done in duplicate. NG and HG = 5 mM and 20 mM D-glucose, respectively; HG/Aza = cells in 20 mM D-glucose for 96 h in the presence of azacytidine; L-Gl = 20 mM L-glucose; ^ = IgG antibody control. ∗p < 0.05 compared to NG and #p < 0.05 compared to HG.

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  Fig. 3. Regulation of DNA methylation and LncCytB expression. LncCytB expression was determined in cells incubated with azacytidine in high glucose by (a) strand-specific PCR using β-actin as a housekeeping gene, and (b) RNA FISH (fuorescein-12-dUTP incorporated LncCytB probe); a representative image captured by Zeiss microscope using a 40X objective and the Apotome module is presented. (c) Arithmetic mean intensity (AMI) of green fluorescence of LncCytB, calculated by Zeiss colocalization software module. Data in the graphs are represented as mean ± SD, obtained from three or more different cell preparations. NG and HG = 5 mM and 20 mM D-glucose, respectively; HG/Aza = 20 mM D-glucose + azacytidine; L-Gl = 20 mM L-glucose. ∗p < 0.05 vs. NG and #p < 0.05 vs. HG.

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  Fig. 4. DNA methylation and mitochondrial genomic stability. (a) Nucleoids were quantified using SYBR gold DNA stain. The images were captured on a ZEISS microscope using a 488 nm wavelength filter and a 40X objective lens. (b) Number of nucleoids were counted, and the values are mean ± SD obtained from 30 to 40 cells in each group. (c). Isolated mitochondria, stained with MitoTraker deep red (MTR) and 1X SYBR green, were scanned under FL1 488 nm and FL3 640 nm wavelength in a flow cytometer. (d) Relative nucleoid contents. Values are represented as mean ± SD obtained from 3 to 4 different cell preparations. NG and HG = 5 mM and 20 mM D-glucose, respectively; HG/Aza = cells in 20 mM D-glucose in the presence of azacytidine; L-Gl = 20 mM L-glucose; Cont = mitochondria without any MTR or SYBR green staining. ∗p < 0.05 compared to NG and #p < 0.05 compared to HG.

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  Fig. 5. Inhibition of DNA methylation and mitochondrial respiration. (a) OCR, (c) basal respiration and (b) maximal respiration rates were determined using a Seahorse XF Cell Mito Stress Test Kit. Each measurement was repeated 3–5 times using five or more wells/group; values in the graphs are presented as mean ± SD. NG = 5 mM D-glucose; HG and HG/Aza = 20 mM D-glucose without and with azacytidine, respectively; L-Gl = 20 mM L-glucose; FCCP = carbonyl cyanide p-trifluoro methoxyphenylhydrazone; Rot/AA = rotenone/antimycin A. ∗p < 0.05 compared to NG and #p < 0.05 compared to HG.

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  Fig. 6. Effect of diabetes on LncCytB DNA methylation. Retina from streptozotocin-induced diabetic rats, with/without azacytidine for eight months was analyzed for DNA methylation by quantifying (a) 5 mC levels using methylated DNA immunoprecipitation method, and (b) MS-PCR in the bisulfite converted DNA. (c) LncCytB transcripts were quantified by strand-specific PCR using β-actin as a housekeeping gene. Each measurement was made in duplicate in 5–7 rats/group. Norm = Normal control, Diab and Diab/Aza = Diabetic rats without or with azacytidine supplementation. ∗p < 0.05 compared to Norm, #p < 0.05 compared to Diab and ^ = IgG antibody control.

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  Fig. 7. Retinal LncCytB DNA methylation in human donors. Retinal (a) 5 mC at LncCytB were quantified in the genomic DNA by DNA immunoprecipitation method using IgG as antibody control (^), and (b) LncCytB transcripts by strand-specific PCR using β-actin as housekeeping gene. Each measurement was performed in duplicate in the retina from 5 or more human donors each with established diabetic retinopathy (DR) or nondiabetic controls (Norm) groups. Data are represented as mean ± SD. ∗p < 0.05 compared with nondiabetic donors.