Background: Diabetic retinopathy (DR) is the leading cause of blindness in the working age population. Transthyretin (TTR) showed a significantly decreased concentration in DR patients and exerted a visual protective effect by repressing neovascularization. This work intended to identify long non coding RNAs (lncRNAs) and explore their potential mechanism underlying the protective role of TTR.Methods: Transcriptome of human retinal endothelial cells (hRECs) treated with low glucose (LG), high glucose (HG) or high glucose with 4 μM TTR (HG + TTR) was conducted. Differentially expressed lncRNAs, mRNAs and TTR related lncRNAs and mRNA were acquired. Functional annotation and Gene Set Enrichment Analysis were applied to analyse TTR affected pathways and processes. Weighted gene co-expression network analysis (WGCNA) was implemented to obtain hub modules and genes. LncRNA-mRNA regulatory networks were constructed based oncis,transand competing endogenous RNAs acting mode. QRT-PCR was conducted to validate the expression of lncRNAs in aqueous humor and serum samples from 30 DR patients and 10 normal controls.Results: RNA-sequencing of hRECs treated with low glucose (LG), high glucose (HG) or high glucose with 4 μM TTR (HG + TTR) was conducted. 146,783 protein-coding transcripts, 12,403 known lncRNA transcripts and 1184 novel non-coding transcripts were characterized. A total of 11,407 differentially expressed mRNAs (DE-mRNAs), 679 differentially expressed lncRNAs (DE-lncRNAs) in HG group versus LG group, 6206 DE-mRNAs and 194 DE-lncRNAs in HG + TTR versus HG group were obtained, respectively. 853 TTR-mRNAs and 48 TTR-lncRNAs were acquired, and functionally involved in cell cycle, apoptosis, inflammation signalling pathway, response to oxidative stress, neovascularization and autophagy. The WGCNA analysis identified a hub module of 133 genes, with the core function of oxidative stress response, angiogenesis, MAPK pathway, cell proliferation and apoptosis. After qRT-PCR validation, a 3-lncRNA regulatory network was proposed. At last, lncRNAsMSTRG.15047.3andAC008403.3showed significantly relative higher expression levels in both aqueous humor and serum samples, compared with normal controls, andFRMD6-AS2was significantly down-regulated.Conclusions: TTR regulated mRNAs and biological processes including oxidative stress, inflammation signalling and autophagy. A 3-lncRNA regulatory network was characterized underlying TTR repressing neovascularization, and showed potential diagnostic performance in DR.

Background/Aims: Diabetic retinopathy (DR) is one of the main causes of blindness in the world. Our previous study showed that transthyretin (TTR) regulates key genes in the Tie2 pathway and inhibits the development of neovascularization in DR, but the mechanism is still unclear. Here, we investigated how TTR affects the progression of neovascularization in DR. Methods: Natural and simulated DR media (hyperglycemia and hypoxia) were used to culture human retinal microvascular endothelial cells (hRECs). Flow cytometry was employed to investigate the effect of TTR on apoptosis of hRECs. Fluorescent labeling and immunofluorescence staining were used to determine the TTR distribution in hRECs. The membrane proteins of hRECs were extracted and applied to a sepharose-TTR column, and the captured proteins were identified by Mass Spectrometric analysis. Gene knock-down and western blotting assays were used to study the key signal pathway of the most abundant identified protein. Results: TTR induced apoptosis of hRECs in an environment that simulated hypoxia. Immunofluorescent staining showed that TTR could enter the nuclei of hRECs. A total of 30 unique TTR-captured proteins were identified by Mass Spectrometry, and glucose-regulated protein 78 (GRP78) was one of the most abundant. Western blotting and gene knock-down indicated that TTR might upregulate GRP78 and facilitate apoptosis through the eIF2α/CHOP pathway. Conclusions: In the DR environment (hyperglycemia and hypoxia), TTR was shown to repress neovascularization by promoting apoptosis of hRECs through a GRP78-dependent pathway.

Background: To investigate the abnormal expression of circ_0008590 and its parent gene, reticuloendotheliosis viral oncogene related B (RELB) in human retinal microvascular endothelial cells (hRECs) in hyperglycemia and the potential mechanism. Methods: The levels of RELB, circ_0008590, and miR-1243 in hRECs or clinical samples were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Dual-luciferase reporter assay was used to test the interaction between RELB/circ_0008590 and miR-1243. Cell Counting Kit-8 (CCK-8), Transwell, flow cytometry (FCM), wound healing, and tube formation assays were used for the physiological investigation. The interaction between human RELB and circ_0008590 was studied in streptozotocin (STZ) induced diabetic retinopathy (DR) C57BL/6 mice. Results: The levels of circ_0008590 and RELB were increased in hRECs in hyperglycemia; during the progression of DR, the levels of circ_0008590 and RELB messenger RNA (mRNA) in aqueous humor were first decreased and then increased, whereas miR-1243 showed an opposite trend. Both RELB 3'-untranslated region (UTR) and circ_0008590 shared a similar binding site for miR-1243. Further, miR-1243 mimic suppressed the proliferation and migration of hRECs, promoting the apoptosis ratio, which could be rescued by the overexpression of circ_0008590. In STZ-induced DR mice, miR-1243 agomir rescued the effects of the overexpression of human RELB. Conclusions: In hyperglycemia, high expression of RELB/circ_0008590 could be suppressed by miR-1243, and the nuclear factor-κB (NF-κB) pathway is subsequently affected.