Regulatory role of miRNA-23a in diabetic retinopathy

Abstract

The present study aimed to investigate the expression of microRNA (miRNA)-23a in blood and tear samples from diabetic retinopathy (DR) patients. Blood and tear samples were obtained from 33 patients with proliferative DR. Additionally, a rat model of DR was established. Reverse transcription-quantitative PCR was used to determine vascular endothelial growth factor (VEGF) mRNA and miRNA-23a expression levels, while ELISA and western blot analysis were performed to determine protein expression levels. Bioinformatics analysis and dual luciferase reporter assay were used to predict and validate the interaction between miRNA-23a and VEGF and cell proliferative ability was assessed with the MTT assay. In comparison to control patients VEGF mRNA and protein expression levels were significantly elevated in the blood and tear samples from patients with DR, while the expression level of miRNA-23a was significantly reduced. In blood and retinal tissues from a rat model of DR, the mRNA and protein expression levels of VEGF were significantly increased, while the miRNA-23a expression level was significantly decreased relative to controls. Dual luciferase reporter assay showed that miRNA-23a bound to the 3'-untranslated region (UTR) of VEGF. Moreover, over-expression of miRNA-23a significantly reduced the expression level of VEGF and the proliferative activity of human retinal microvascular endothelial cells. The elevated VEGF expression in the blood and tears of patients with DR may be related to the reduced miRNA-23a expression. miRNA-23a may regulate microvascular growth at the retina via VEGF and contribute to DR progression.

Keywords: diabetic retinopathy; miRNA-23a; vascular endothelial growth factor.

Figure 1

Analyses of VEGF mRNA and protein expression levels. VEGF mRNA expression levels in the (A) serum and (B) tear samples from the PDR were assessed using RT-qPCR. The VEGF concentration in the (C) serum and (D) tear samples from the PDR were determined by ELISA. The VEGF mRNA expression levels in the (E) serum and (F) retina tissues from the rat DM were determined with RT-qPCR. The VEGF concentration in the (G) serum and (H) retina tissues from the rat DM were determined with ELISA and western blot analysis, respectively. ^*P<0.05, ^**P<0.01 vs. control. VEGF, vascular endothelial growth factor; RT-qPCR, reverse transcription quantitative PCR; Ctrl, control; PDR, patients with diabetic retinopathy; DM, diabetic model.

Figure 2

Analyses of miRNA-23a expression levels. The miRNA-23a expression levels in the (A) serum and (B) tear samples from the PDR were determined with RT-qPCR. The miRNA-23a expression levels in the (C) serum and (D) retina tissues from the rat DM were determined with RT-qPCR. ^*P<0.05, ^**P<0.01 vs. control. miR, microRNA; PDR, patients with diabetic retinopathy; RT-qPCR, reverse transcription quantitative PCR; DM, diabetic model.

Figure 3

Bioinformatics prediction of VEGF regulation. (A) Bioinformatics prediction of up-stream regulating gene of VEGF. (B) Dual luciferase reporter assay, the 293T cells were co-transfected with the agomiRNA-23a and pmiR-REPORT luciferase reporter plasmids containing the wild-type and mutant sequences, respectively. The luciferase values were detected and analyzed. ^**P<0.01 vs. NC. VEGF, vascular endothelial growth factor; miR, microRNA; NC, normal control.

Figure 4

Effects of agomiRNA-23a transfection on hRMECs. hRMECs were transfected with agomiRNA-23a, and the expression levels of (A) miRNA-23a and (B) VEGF were determined with reverse transcription-quantitative-PCR. The VEGF protein expression levels were determined in the transfected hRMECs with (C) western blot analysis. (D) After transfection, cell proliferative ability was assessed with the MTT assay. ^*P<0.05, ^**P<0.01 vs. NC. miR, microRNA; hRMEC, human retinal microvascular endothelial cells; NC, normal control.