Long non-coding ribonucleic acid urothelial carcinoma-associated 1 promotes high glucose-induced human retinal endothelial cells angiogenesis through regulating micro-ribonucleic acid-624-3p/vascular endothelial growth factor C

Abstract

Aims/introduction: Emerging evidence has indicated that long non-coding ribonucleic acids play important roles in the development and progression of diabetic retinopathy (DR). It is reported that urothelial carcinoma-associated 1 (UCA1) is highly expressed in diabetic lymphoendothelial cells and influences glucose metabolism in rats with DR. The aim of the present study was to explore the role of UCA1 in the mechanism of DR.

Materials and methods: Gene expression analyses in fibrovascular membranes excised from patients with DR using public microarray datasets (GSE60436). Reverse transcription polymerase chain reaction was carried out to detect UCA1, micro-ribonucleic acid (miR)-624-3p and vascular endothelial growth factor C (VEGF-C) expressions in the blood of patients and human retinal endothelial cells (HRECs). Furthermore, Cell Counting kit-8, Transwell assay, and tube formation assay were used to identify biological effects of UCA1 on HRECs proliferation, migration ability and angiogenesis in vitro.

Results: UCA1 and VEGF-C were elevated in DR patients and high glucose-induced HRECs cell lines, whereas miR-624-3p was decreased. UCA1 inhibition inhibited proliferation, angiogenesis and migration of HRECs cells under high-glucose condition. Luciferase reporter assay showed that UCA1 could sponge with miR-624-3p, which could directly target VEGF-C. Finally, we proved a pathway that UCA1 promoted cell proliferation, migration and angiogenesis through sponging with miR-624-3p, thereby upregulating VEGF-C in high-glucose-induced HRECs.

Conclusions: We identified UCA1 as an important factor associated with DR, which could regulate the expression of VEGF-C by sponging miR-624-3p in human retinal endothelial cells. Our results pave the way for further studies on diagnostic and therapeutic studies related to UCA1 in DR patients.

Keywords: Angiogenesis; Diabetic retinopathy; Urothelial carcinoma-associated 1.

Figure 1

Identification of urothelial carcinoma‐associated 1 (UCA1), miR‐624‐3p and vascular endothelial growth factor C (VEGF‐C) in diabetic retinopathy (DR) and human retinal endothelial cells. (a) Clustered heatmap of the differentially expressed genes in DR. Upregulated genes are shown in red and downregulated genes are shown in green. (b) Volcano plots comparing gene expression between DR patients and controls. The red dots represent the significantly differentially expressed genes (|logFC|>1 and P < 0.05). (c–e) Reverse transcription polymerase chain reaction was used to detect UCA1,VEGF‐C and miR‐624‐3p levels in blood from DR patients (n = 50), type 2 diabetes (T2D) patients (n = 50) and non‐DR individuals (n = 50). (f) Reverse transcription polymerase chain reaction was carried out to measure the expression level of UCA1,VEGF‐C and micro‐ribonucleic acid (miR)‐624‐3p in human retinal endothelial cells treated with 5 mmol/L or 25 mmol/L glucose for 48 h. All values represent the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Urothelial carcinoma‐associated 1 (UCA1) promotes high‐glucose‐induced cell proliferation, migration and angiogenesis. (a) Lactate dehydrogenase (LDH) analysis of human retinal endothelial cells of small interfering si‐UCA1group and si‐NC group, (b) UCA1 expressions were detected by reverse transcription polymerase chain reaction. (c) Proliferation abilities of human retinal endothelial cells by Cell Counting Kit‐8 assay. (d) Migration abilities were measured by Transwell assay. (e) Tube formation assay was carried out to detect the angiogenesis ability of human retinal endothelial cells. si‐UCA1: small RNA interfering group; si‐NC: without small interfering group; ***P < 0.001.

Figure 3

Urothelial carcinoma‐associated 1 (UCA1) acted as a sponge of miR‐624‐3p. (a) Predicted miR‐624‐3p binding sites in UCA1 and luciferase assays in wild‐type (WT)‐UCA1 or mutant (MUT)‐UCA1 human retinal endothelial cells co‐transfected with micro‐ribonucleic acid (miR)‐NC or miR‐624‐3p. (b) The pull‐down experiments show that the anti‐Argonaute 2 (AGO2) antibody significantly enriched UCA1. (c) The enrichment of UCA1 in ribonucleic acid (RNA) immunoprecipitation. (d) miR‐624‐3p expression in human retinal endothelial cells transfected with small interfering (si)‐UCA1 or overexpression (oeUCA1). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase. si‐UCA1: small RNA interfering group; si‐NC: without small interfering group.

Figure 4

Urothelial carcinoma‐associated 1 (UCA1) promoted high‐glucose‐induced human retinal endothelial cells proliferation, migration and angiogenesis through regulating micro‐ribonucleic acid (miR)‐624‐3p/vascular endothelial growth factor V (VEGF‐C). (a) Schematic showing the predicted miR‐624‐3p sites in VEGF‐C and luciferase assays in wild‐type (WT)‐VEGF‐C or mutant (MUT)‐VEGF‐C human retinal endothelial cells co‐transfected with miR‐NC or miR‐624‐3p. (b) The messenger ribonucleic acid expressions of VEGF‐C were detected by reverse transcription polymerase chain reaction. (c) Protein levels of VEGF‐C were detected by western blot. (d) Cell Counting Kit‐8 assay was used to measure cell proliferation abilities. (e) Migration abilities were measured by Transwell assay. (f) Tube formation assay was carried out to detect cell angiogenesis ability. si‐UCA1: small RNA interfering group; si‐NC: without small interfering group. *P < 0.05, **P < 0.01, ***P < 0.001.