Abstract
Purpose :
Diabetes-induced metabolic dysfunction leads to the dysfunction of retinal cells, thus causing diabetic retinopathy (DR). The RPE is critical in visual function and provides crucial metabolic support for retinal homeostasis. MicroRNAs can simultaneously regulate multiple gene products thus having pivotal roles in disease pathogenesis. Since miR182-5p is involved in the regulation of glycolysis and angiogenesis, two important aspects related to DR, we investigated the expression of miRNA182-5p in proliferative diabetic retinopathy (PDR) patients and in a model hyperglycemic stress in human RPE cells.
Methods :
Vitreous samples were collected from the vitrectomy patients with prior approval of the Institutional Ethics Committee and written informed consent. Total RNA was extracted from patient vitreous samples followed by analysis of miR182-5p and its target genes using qPCR from PDR (n=48) and macular hole (n=22) subjects. ARPE-19 cells, cultured in DMEM under differential glucose conditions (5mM and 25mM) were used for mRNA(qPCR) and protein (immunoblotting) expression, metabolic (Seahorse XF) and biochemical assays. ARPE19 cells were transfected with miRNA182 mimic or its antagomir to study the gain and loss of function effects. Secreted VEGF levels were estimated by ELISA.
Results :
We found significantly increased miR182-5p levels (p<0.05) in vitreous humor samples from patients with advanced stages of PDR. The RPE cells under hyperglycemic stress showed similar elevation in miR182-5p expression with altered glycolytic pathway drivers such as HK2, PFKP and PKM2. Additionally, hyperglycemic RPE cells exhibited reduced FOXO1 and enhanced Akt activation. Similarly, cells transfected with miR182-5p mimic phenocopied the enhanced basal and compensatory glycolytic rates(p<0.05) observed in hyperglycemia with increased VEGF secretion(p<0.05). Conversely, inhibiting miR182-5p reduced Akt activation, glycolytic pathway proteins, and VEGF while stabilizing FOXO1.
Conclusions :
Glycolysis-associated proteins downstream of the FoxO1-Akt axis were regulated by miR182-5p. Further, miR182-5p also led to the increased expression of VEGFR2 and VEGF levels, likely via inhibition of ZNF24. Thus, the FoxO1-Akt-glycolysis/VEGF pathway, which drives metabolic dysfunction, and the concurrent angiogenic signaling in PDR may be potentially targeted for treatment via miR182-5p.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.