Dicer 1 loss and increased Alu RNA in the pathogenesis of diabetic retinopathy

Abstract

Purpose: Endothelial dysfunction and loss of vascular repair by dysfunctional endothelial progenitor cells (EPCs) play a central role in the pathogenesis of diabetic retinopathy. The expression of dicer, the endoribonuclease required for processing of miRNA and regulating of Alu RNA, was examined in freshly isolated peripheral blood CD34+ cells and in the in vitro expanded progenitor population, endothelial colony forming cells (ECFCs) from human subjects with and without diabetic retinopathy and in control subjects. We also examined dicer mRNA expression in the retina and EPCs of db/db diabetic mice and db/m controls.

Methods: Diabetic and control mice were maintained under a 12h:12h/light:dark cycle and euthanized every 4 hrs. RT-PCR was used to determine retinal dicer mRNA levels from controls and diabetics. ELISA was used to determine dicer protein levels in CD34+ cells from diabetic (n=10) and control human subjects (n=10). Endothelial colony forming cells (ECFCs), an in vitro expanded endothelial progenitor population, were derived from diabetic and control human subjects. ECFCs were synchronized by 50% horse serum shock and then harvested to assess diurnal DICER mRNA levels. The effect of Alu double stranded RNA on apoptosis and cell proliferation in human retinal endothelial cells (HRECs) and EPCs was examined by the caspase 3 activity assay and colorimetric cell proliferation assay.

Results: Retinal dicer mRNA levels exhibited a diurnal oscillation in mice with peak expression occurring at ZT21 and the nadir at ZT9. Dicer mRNA expression was significantly reduced in the retina of diabetic mice at 8 weeks post onset of diabetes. Dicer protein expression in diabetic human CD34+ progenitor cells was reduced by 41% when compared to that from non-diabetic controls. Diabetic ECFCs lost the diurnal pattern of dicer expression observed in control nondiabetic ECFCs. Overexpression of Alu dsRNA reduced cell proliferation and increased caspase 3 activity in HRECs and EPCs.

Conclusions: Dicer controls miRNA processing and is also involved in the removal of toxic Alu dsRNAs. Diabetic retina and EPCs show both decreased levels of dicer and loss of diurnal dicer expression compared to nondiabetic tissues. Our results suggest that the reduction of dicer expression and loss of diurnal rhythm of dicer in key tissues such as the retina and in progenitor cells of diabetics could contribute to the pathogenesis of diabetic retinopathy

Keywords: 499 diabetic retinopathy • 688 retina • 721 stem cells