Regulatory Effects of Mir365 and Bdnf in Müller Cells Involved in Diabetic Retina

Abstract

Purpose: To examine the inhibitory role of Brain-derived neurotrophic factor (BDNF) on neuro protection in diabetic retinas, and to study this regulatory mechanism by microRNA (miRNA) at post-transcriptional level in vivo.

Methods: miR365 and its target, bdnf, was predicted by bioinformatics analysis and validated by luciferase assay in vitro. The interaction between the two was detected in rat Müller cells (rMC-1 cell line) by overexpressing or inhibiting mir365 level. Glyoxal (AGEs stress) was used to treat rat Müller cells (rMC-1 cell line), and then the mRNA level of bdnf and mir365 were detected. Further, the level of miRNA of interest in diabetic retina was detected by qRT-PCR. Neurosensory retina tissues were collected at different time points from diabetes onset in STZ (Streptozotocin)-induced diabetic rats ex vivo. The differential expression level of bdnf was validated by qRT-PCR and ELISA. In vivo, a virus based anti-mir365 was delivered into diabetic rats and its protection effect was detected.

Results: bdnf is one of the important targets of Mir365, and their interaction was confirmed by in vitro double luciferase activity assay in HEK293 cell and negative regulation in rMC-1. In 2mM glyoxal treated rMC-1(AGEs stress), the expression of mir365 was enhanced and bdnf was decreased after treatment. In diabetic retina, the levels of mir365 increased in 1 week and 2 week diabetes, while bdnf expression level showed significant decreased from 4 week diabetes onset as compared to normal controls. After anti-mir365 treatment in diabetic rats, the apoptosis in neuro retina was attenuated.

Conclusions: In diabetic retinas, as mir365 expression was badly promoted, one of its important targets, bdnf expression level significantly down-regulated from 4 weeks after diabetes onset. Since neuro protection is one of the most important need in clinic diabetic retinopathy patient, our in vivo result that anti-Mir365 treatment in rat can protect neuro retina from apoptosis is somehow meaningful to the treatment of DR. also miRNAs, as a new molecular therapy agent, show its potent application in near future.

Keywords: 499 diabetic retinopathy • 615 neuroprotection • 426 apoptosis/cell death