Mitochondrial Fragmentation and Long Noncoding RNA MALAT1 in Diabetic Retinopathy

Figures

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  Figure 1. Effect of MALAT1-siRNA on the glucose-induced downregulation/inhibition of Mfn2. HRECs incubated in high glucose for 96 h were analyzed for MALAT1 (a) transcripts by qRT-PCR using β-actin as a housekeeping gene and (b) expression by RNA FISH. (c) AMI of MALAT1, calculated from 25–30 cells in each group. Mfn2 (d) gene transcripts were quantified by qRT-PCR by employing β-actin as a housekeeping gene, and (e) GTPase activity was examined by quantifying phosphate released at 620 nm. Using values from cells in normal glucose as 100%, the percentage change in the phosphate released was calculated. (f) Transfection efficiency of MALAT1-siRNA. The values in the graphs are represented as mean ± SD, obtained from 3–4 cell preparations, with each measurement made in duplicate/triplicate. NG and HG = 5 mM or 20 mM D-glucose; HG/MAL-si and HG/SC = MALAT1-siRNA or scrambled control RNA-transfected HRECs in HG, L-Gl = 20 mM L-glucose. * p < 0.05 compared with NG and # p < 0.05 compared with HG.

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  Figure 2. MALAT1 upregulation and Mfn2 promoter DNA methylation in HRECs. Using IgG as an antibody control, (a) 5mC levels at Mfn2 promoter DNA were quantified by the methylated DNA capture method, and (b) Dnmt1 binding at the Mfn2 promoter was measured by the ChIP technique. MALAT1 binding at (c) the Mfn2 promoter was determined by the ChIRP technique, and (d) Dnmt1 by the RIP technique. NG and HG = cells in 5 mM or 20 mM D-glucose; HG/MAL-si and HG/SC = MALAT1-siRNA or scrambled control RNA-transfected cells in HG, L-Gl = 20 mM L-glucose. ^ = IgG antibody control, * p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 3. Mitochondrial fragmentation and ultrastructure: (a) Mitochondrial fragmentation was evaluated by live cell imaging using an ApoTome fluorescence microscope and a 63X objective in HRECs stained with MitoTracker green. The red box area is zoomed, and the outlines are plotted using ImageJ software, version 1.53K. (b,c) Aspect ratio and form factor were calculated by ImageJ software in fluorescent images converted to 8-bit images. Values are mean ± SD obtained from 10 to 12 images/conditions with 3–4 cells/image. (d) Representative electron micrographs of cells; red arrows point to the mitochondria; (e) mitochondrial length was measured using ImageJ software, and the values are represented as mean ± SD from 10 to 12 images/experimental conditions with 8–10 mitochondria/image. NG and HG = 5 mM or 20 mM D-glucose; HG/MAL-si and HG/SC = MALAT1-siRNA or scrambled RNA-transfected cells in HG, L-Gl = 20 mM L-glucose.* p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 4. Effect of the protection of Mfn2 downregulation on mitochondrial damage. (a) Oxygen consumption was measured by a Seahorse XF Analyzer Cell Mito Stress Test Kit. Each measurement was repeated 3–5 times using four or more wells/group, and the values are presented as mean ± SD from 3 to 4 HREC preparations. (b–d) represent basal respiration, maximal respiration, and spare respiratory capacity, respectively. (e) Representative image showing retinal endothelial cells stained with JC-1, and (f) the ratio of J-aggregates (red) and J-monomers (green). NG and HG = cells in 5 mM or 20 mM D-glucose; HG/MAL-si and HG/SC = MALAT1-siRNA or scrambled control RNA-transfected cells in HG, L-Gl = 20 mM L-glucose.* p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 5. Effect of MALAT1 regulation on Mfn2 in retinal Muller cells. RMCs, incubated in high glucose for 96 hrs, were analyzed for MALAT1 (a) transcripts by qRT-PCR using β-actin as a housekeeping gene, and (b) expression was analyzed by RNA-FISH. (c) Mean AMI of MALAT1, calculated from >20 cells/coverslip. (d) Mfn2 gene transcripts, quantified by qRT-PCR, with β-actin as a housekeeping gene. (e) Transfection efficiency of MALAT1. The values mean ± SD from 3 cell preparations. NG and HG = 5 mM and 20 mM D-glucose, respectively; HG/MAL-si and HG/SC = MALAT1-siRNA or scrambled RNA-transfected cells in HG; L-Gl = 20 mM L-glucose * p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 6. MALAT1 upregulation and Mfn2 promoter DNA methylation in RMCs. (a) The 5mC levels at the Mfn2 promoter DNA were quantified by the Methylated DNA capture technique, and (b) Dnmt1 binding at Mfn2 was quantified by the ChIP technique. MALAT1 binding at (c) the Mfn2 promoter was determined by the ChIRP technique, and (d) Dnmt1 by the RIP technique. NG = 5 mM D-glucose; HG = 20 mM D-glucose; HG/MAL-si and HG/SC = MALAT1-siRNA or scrambled RNA-transfected cells in HG, 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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  Figure 7. Effect of MALAT1-siRNA on mitochondrial fragmentation: (a) Using live cell imaging, RMCs stained with MitoTracker green were visualized using an Zeiss ApoTome fluorescence microscope (Carl Zeiss Inc., Baden-Wurttemberg, Germany)and a 63X objective. The zoomed area in the red box was used to plot the outlines. (b,c) Aspect ratio and form factor were measured by ImageJ software, and the values are mean ± SD from 10 to 12 images/condition with 3–4 cells/image. NG = 5 mM D-glucose; HG and HG/MAL-si = untransfected or MALAT1-siRNA-transfected cells in 20 mM D-glucose.* p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 8. Working model showing the effect of MALAT1 on the Mfn2 expression development of diabetic retinopathy.