Abstract
Purpose :
Although diabetic retinopathy (DR) is the most common microvascular complication in diabetes, neuro-retinal degeneration is observed in diabetes patient without any signs of DR. However, little is known about the mechanisms that leads to loss of neuro-retinal cell before generating vascular complication in diabetes. In this study, we investigated the neuronal RNA binding protein HuD as a novel regulator responsible for neuro-retinal degeneration at the early stage of diabetes.
Methods :
The expressions of RNA and proteins in R-28 cells and rat retinal tissues (N=3) by RT-qPCR, as well as Western blotting and immunohistochemistry (IHC), respectively. Association between HuD and Cryaa mRNA were analyzed using ribonucleoprotein complex-immunoprecipitation (RNP-IP) and EGFP-reporter assay was carried out to verify regulation of Cryaa mRNA by HuD. The cell viability was analyzed by MTT (3-(4,5-Dimethylthiazol-2-yl) assay.
Results :
We firstly identified the expression of HuD and alpha-crystallin A (CRYAA) in ganglion cell in retina. HuD and CRYAA was down-regulated in retina streptozotocin (STZ)-induced diabetic rat and also in neuro-retinal cell lines (R-28) treated with high glucose. We identified Cryaa mRNA as a novel target transcript of HuD, and also demonstrated that HuD post-transcriptionally regulates the expression of Cryaa mRNA by binding to its 3’-untraslated region (UTR). HuD silencing and overexpression positively regulated the expressions of Cryaa mRNA and proteins. We demonstrated that increases of inflammatory cytokines, IL-1b, IL-6, Tnfα, in R-28 cells by high glucose were due to both CRYAA and HuD. silencing of HuD and Cryaa enhanced high glucose-induced R-28 cell death, whereas overexpression of them alleviated that.
Conclusions :
HuD post-transcriptionally regulates CRYAA expression, which involved in function and viability of neuro-retinal cell under diabetic condition. Our results suggest that HuD play a crucial role in neuro-retinal cells and has potential roles as a prognostic factor and therapeutic target for diabetic neuro-retinal degeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.