MiR-200b downregulates Oxidation resistance 1 (Oxr1) in diabetic retinopathy of a type 1 diabetic animal model

Abstract

Purpose: Diabetic retinopathy is a microvascular complication of diabetes mellitus and is the leading cause of blindness in developed countries. MicroRNAs (miRNAs) are small, no-coding RNAs which negatively regulate target gene expression at the post-transcription level. miRNAs play important roles in pathological processes of many diseases. This study is to investigate changes of the miRNA profile in diabetic retinopathy and to elucidate the potential role of a specific miRNA, miR-200b in a type 1 diabetic mouse model.

Methods: The visual functions of both Akita and wild-type (WT) mice were examined by electroretinogram (ERG) recording. RNAs were extracted from the retina of Akita mice (9 months of age) and age-matched WT mice. miRNA expression profiles were compared using a miRNA-specific microarray, and the expression change of miR-200b was confirmed by Taqman qPCR. The level of miR-200b was also examined in a human Müller cell line (MIO-M1) after the treatment of 4-hydroxynonenal (4-HNE). The target gene(s) of miR-200b was predicted in silico and confirmed by luciferase assay. The function and role of target gene(s) of miR-200b was tested and verified in MIO-M1 cells by Western blotting and TUNEL assay.

Results: ERG recording showed progressive declines of ERG responses in Akita mice when compared with age-matched WT mice. Microarray and real-time PCR indicated that miR-200b was significantly up-regulated in the retina of Akita mice. Computational sequence analysis and luciferase assay identified that Oxidation resistant 1 (Oxr1) was a direct target gene of miR-200b. In the retina of Akita mice and in MIO-M1 cells exposed to 4-HNE, the up-regulation of miR-200b correlated with down-regulation of the Oxr1 mRNA levels in the retina. In MIO-M1 cells, miR-200b mimic transfection down-regulated Oxr1 expression, while the inhibitor of miR-200b increased Oxr1 expression. Moreover, the over-expression of recombinant Oxr1 enhanced cell survival and reduced the 3-nitrotyrosine levels in MIO-M1 cells exposed to 4-HNE.

Conclusions: The up-regulation of miR-200b in diabetes reduces the expression of Oxr1 and weakens the anti-oxidant function of Oxr1 in diabetic retinopathy.

Keywords: 499 diabetic retinopathy