Role of Hsa_circ_0000880 in the Regulation of High Glucose-Induced Apoptosis of Retinal Microvascular Endothelial Cells

Abstract

Purpose: Circular RNAs (circRNAs) have been verified to participate in multiple biological processes and disease progression. Yet, the role of circRNAs in the pathogenesis of diabetic retinopathy (DR) is still poorly understood and deserves further study. This study aimed to investigate the role of circRNAs in the regulation of high glucose (HG)-induced apoptosis of retinal microvascular endothelial cells (RMECs).

Methods: Epiretinal membranes from patients with DR and nondiabetic patients with idiopathic macular epiretinal membrane were collected for this study. The circRNA microarrays were performed using high-throughput sequencing. Hierarchical clustering, functional enrichment, and network regulation analyses were used to analyze the data generated by high-throughput sequencing. Next, RMECs were subjected to HG (25 mM) conditions to induce RMECs apoptosis in vitro. A series of experiments, such as Transwell, the Scratch wound, and tube formation, were conducted to explore the regulatory effect of circRNA on RMECs. Fluorescence in situ hybridization (FISH), immunofluorescence staining, and Western blot were used to study the mechanism underlying circRNA-mediated regulation.

Results: A total of 53 differentially expressed circRNAs were found in patients with DR. Among these, hsa_circ_0000880 was significantly upregulated in both the diabetic epiretinal membranes and in an in vitro DR model of HG-treated RMECs. Hsa_circ_0000880 knockout facilitated RMECs vitality and decreased the paracellular permeability of RMECs under hyperglycemia. More importantly, silencing of hsa_circ_0000880 significantly inhibited HG-induced ROS production and RMECs apoptosis. Hsa_circ_0000880 acted as an endogenous sponge for eukaryotic initiation factor 4A-III (EIF4A3). Knockout of hsa_circ_0000880 reversed HG-induced decrease in EIF4A3 protein level.

Conclusions: Our findings suggest that hsa_circ_0000880 is a novel circRNA can induce RMECs apoptosis in response to HG conditions by sponging EIF4A3, offering an innovative treatment approach against DR.

Translational relevance: The circRNAs participate in the dysregulation of microvascular endothelial function induced by HG conditions, indicating a promising therapeutic target for DR.

Figure 1.

Identification of differentially expressed circRNAs in the epiretinal membranes between patients with PDR and nondiabetic patients. (A) The heatmap was constructed to compare expression of circRNAs between patients with PDR and nondiabetic patients. High expression is indicated in red and low expression is indicated in green. (B, C) GO annotation enrichment analysis and KEGG pathway analysis of the upregulated circRNAs. The vertical axis is the description of GO terms and the horizontal axis is the enrichment score (-log10 [P value]) of the pathways. (D) Validation of hsa_circ_0000880 by qRT-PCR in the epiretinal membranes between patients with PDR and nondiabetic patients. (E) The qRT-PCR analysis of three circRNAs expression in the vitro model of hyperglycemia-induced RMECs injury. The relative expression levels were normalized to the expression of GAPDH. (F) CircRNA-miRNA interactions and circRNA-protein interactions were constructed using Cytoscape. The light blue circles represent miRNAs. The red triangles represent differentially expressed circRNAs. The red hexagons represent circRNA-targeted proteins. The yellow square represents EIF4A3. The values are expressed as the means ± SEMs. **P < 0.01, ***P < 0.001. PDR, proliferative diabetic retinopathy; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 2.

The effect of hsa_circ_0000880 silencing on the viability of RMECs. (A) RMECs were transiently transfected with hsa_circ_0000880 siRNA for 24 hours. (B) Relative expression level of has_circ_0000880 in the NG, the HG, and the HG+Si-circRNA groups. (C) MTT assay showed the cell viability in the NG, the HG, and the HG+Si-circRNA groups. (D) Representative image of RMECs after siRNA transfection. The values are expressed as the means ± SEMs. **P < 0.01. RMECs, retinal microvascular endothelial cells; HG, high glucose.

Figure 3.

The effect of hsa_circ_0000880 on the wound healing and cell migration of RMECs under hyperglycemic conditions. (A) The scratch wound assay in the NG, the HG, the HG+Si-circRNA, and the HG+Si-NC groups (scale bar = 200 µm). (B) Transwell assay in the NG, the HG, the HG+Si-circRNA, and the HG+Si-NC groups (scale bar = 50 µm). (C) The migration area in the scratch wound assay. (D) The number of migration cells per field in the Transwell experiment. (E) Relative migration rate in the Transwell experiment. The values are expressed as the means ± SEMs. **P < 0.01, ***P < 0.001. RMECs, retinal microvascular endothelial cells; HG, high glucose.

Figure 4.

The effect of hsa_circ_0000880 on the tube formation of RMECs under hyperglycemic conditions. (A) Tube networks formation of RMECs in the NG, the HG, the HG+Si-circRNA, and the HG+Si-NC groups. (B) Number of branch points in the NG, the HG, the HG+Si-circRNA, and the HG+Si-NC groups. (C) Number of tube-like structures in the NG, the HG, the HG+Si-circRNA, and the HG+Si-NC groups. The values are expressed as the means ± SEMs. *P < 0.05. RMECs, retinal microvascular endothelial cells; HG, high glucose. Scale bar = 200 µm.

Figure 5.

Hsa_circ_0000880 silencing decreased the production of HG-induced ROS and suppressed the apoptosis of RMECs under HG conditions. (A, C) Cell apoptosis was determined by TUNEL assay. (B, D) Intracellular ROS quantities were assessed using DCFH-DA staining. The values are expressed as the means ± SEMs. **P < 0.01, ***P < 0.001. HG, high glucose; ROS, reactive oxygen species; RMECs, retinal microvascular endothelial cells; DCFH-DA, dichlorodihydrofluorescein diacetate; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling. Scale bar = 50 µm.

Figure 6.

Hsa_circ_0000880 localized in the nucleus and interact with EIF4A3. (A) Mature sequence of hsa_circ_0000880. (B) Matching flanking regions for hsa_circ_0000880-EIF4A3 binding. (C) FISH images showing that hsa_circ_0000880 was mostly located in the nucleus and co-localized with EIF4A3 in RMECs. (D, E) The relative protein expression of EIF4A3 in HG-treated and hsa_circ_0000880 knockdown RMECs. The values are expressed as the means ± SEMs. *P < 0.05, ***P < 0.001. RMECs, retinal microvascular endothelial cells; EIF4A3, eukaryotic initiation factor 4A-III; FISH, Fluorescence in situ hybridization assay.