Nuclear Genome-Encoded Long Noncoding RNAs and Mitochondrial Damage in Diabetic Retinopathy

Figures

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  Figure 1. Heatmap analysis of LncRNAs in HRECs incubated in high glucose. RNA sequencing was performed in the nuclear fraction of the cells incubated in normal and high glucose; the figure represents a heatmap of two different cell preparations, each incubated in 5 mM d-glucose (NG1 and NG2) and in 20 mM d-glucose (HG1 and HG2).

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  Figure 2. Validation of differently expressed LncRNAs in high glucose. Nine differentially expressed LncRNAs from the heatmap were quantified by qRT-PCR using β-actin as the housekeeping gene. Each measurement was made in duplicate in three different cell preparations. Values obtained from cells in NG were considered as 1, and are represented as mean ± SD. * p < 0.05 vs. NG.

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  Figure 3. Effect of high glucose on mitochondrial localization of LncMALAT1 and LncNEAT1. (a,c) Using RNA-FISH technique, cells fixed with 4% paraformaldehyde were incubated with fluorescein-labelled denatured probes. Mitochondrial localization was performed using CoxIV antibody and Texas Red-conjugated secondary antibody. Cells were mounted using DAPI (blue)-containing mounting medium. (b,d) Signals of fluorescein-12-dUTP incorporated probe (green) were visualized to determine the hybridized probes. Pearson’s correlation coefficient of CoxIV with LncMALAT1 or LncNEAT1 was calculated by using the “Region of Interest” in the outer nuclear region from 25 or more cells in each group. Pearson’s correlation coefficient values in the graphs are represented as mean ± SD. NG and HG = Cells in 5 mM or 20 mM d-glucose; L-Gl = cells in 20 mM l-glucose; * p < 0.05 vs. NG.

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  Figure 4. Effect of inhibition of LncMALAT1 or LncNEAT1 on high glucose-induced mitochondrial ROS levels. (a) Live cells, incubated with MitoSox red and MitoTracker green, were imaged under a Zeiss Apotome at 40× objective. (b) Mean MitoSox intensity, obtained from 30–50 cells, in each incubation condition. (c,d) Transfection efficiency of MALAT1 and NEAT1, as determined by their respective gene transcripts. Each measurement was made in duplicate in 2–3 different cell preparations. NG and HG = Cells in 5 mM or 20 mM d-glucose, MAL-si and NEAT-si = Cells transfected with MALAT1-siRNA or NEAT1-siRNA and incubated in high glucose, SC = Cells transfected with scrambled control RNA and incubated in high glucose, NG/MAL-si and NG/NEA-si = cells transfected with MALAT1-siRNA or NEAT1-siRNA in normal glucose, and L-Gl = Cells incubated in 20 mM l-glucose. * p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 5. Silencing of MALT1 or NEAT1 on mitochondrial membrane integrity. (a) Representative image obtained using cationic dye JC-1 to determine mitochondrial membrane potential. Cells were visualized under a Zeiss ApoTome fluorescence microscope at 20× objective. Green fluorescence represents the depolarized (monomer) mitochondria and orange represents hyperpolarized (J aggregates) mitochondria. (b) The graph represents the ratio of intensity of red and green fluorescence. * p < 0.05 vs. NG and # p < 0.05 vs. HG.

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  Figure 6. Mitochondrial DNA damage and or LncNEAT1. (a) Mitochondria DNA damage was quantified by extended-length PCR using long mtDNA (8.8 kbp) and short (117 bp) amplicons of the mtDNA. (b) Cytb gene transcripts were quantified by qRT-PCR using β-actin as the housekeeping gene. Each measurement was made in duplicate/triplicate in 3–4 different cell preparations, and the histograms represent values as mean ± SD. NG = 5 mM d-glucose; HG = 20 mM d-glucose, MAL-si, NEAT-si and SC = Cells transfected with MALAT1-siRNA, NEAT1-siRNA, or scrambled control RNA respectively, and incubated in 20 mM d-glucose, L-Gl = cells in 20 mM l-glucose. * and # p < 0.05 vs. NG and HG respectively.

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  Figure 7. Effect of silencing of MALAT1 or NEAT1 on nucleoids in mtDNA: (a) Nucleoids were quantified by staining cells using SYBR (green) and imaging at 40× objective. (b) The histogram represents the number of nucleoids from 5 to 8 images/group, quantified using the ZEISS analysis module. (c) Representative image of the localization of nucleoids in the mitochondria using SYBR (green) and mitochondrial marker Cytb (Cytb, red) staining. (d) The images were calibrated with the ZEISS proinbuilt software package, and using random region of interest in the area outside the nucleus, Pearson’s correlation coefficient between red and green stain was determined. Each measurement was made in duplicate in 3–4 different cell preparations. NG = 5 mM d-glucose; HG = 20 mM d-glucose, MAL-si, NEAT-si and SC = Cells transfected with MALAT1-siRNA, NEAT1-siRNA, or scrambled control RNA, respectively, and incubated in 20 mM d-glucose, L-Gl = cells in 20 mM l-glucose. * p < 0.05 vs. NG and # p < 0.05 vs. HG.