Glyoxal-induced Small Non-coding Rnas From Rat Muller Cells Involve In The Pathogenesis Of Early Diabetic Retinopathy (dr)

Abstract

Purpose: : To profile small non-coding RNAs (sncRNA) in rat Muller cells under oxidative stress and then to investigate their possible role in the pathogenesis of diabetic retinopathy.

Methods: : sncRNA library from glyoxal-induced Muller cell line was constructed by denaturing PAGE-based size fractionation. Recombinant clones were randomly sequenced. Bioinformatics analysis was performed to identify the known and novel miRNAs. Putative novel miRNAs were validated by both quantitative RT-PCR and Northern blotting. Double luciferase assay was carried out to confirm the interaction between target genes and miRNAs. The expression of miRNA of interest was examined in STZ-induced DR by qRT-PCR.

Results: : Totally 369 small RNAs sequences were analyzed, 16% of sncRNAs were known miRNAs, 11% of them were not annotated in the database,1 of which was proved to be a novel miRNA from 28s RNA. Using Targetscan analysis, 3’UTR of a cytokine mRNA was predicted to be the target of mir-25 and double luciferase activity assay demonstrated their interaction. Western blot is needed for further confirmation. In retinas of STZ-induced DR rats, the expression of mir-25 increased significantly in 1w and 2w groups after diabetes onset (p<0.01),but it decreased in 4w and 6w groups with no statistical significance compared to the control, while its target gene mRNA showed a downregulation in the 2w group (p<0.05). Mir-25 upregualtion in diabetic retina preceded its’target mRNA downregualtion. Mir-31 expression, whose target is VEGF, showed a decreased tendency in 1w, 2w, and 4w groups (p>0.05), while it was downregulated significantly in the 6w group(p<0.01) as compared to control.

Conclusions: : Oxidative stress-induced microRNAs from rMC, such as mir-25 and mir31 may be involved in the pathogenesis of early DR by regulating vital molecules such as VEGF. Meanwhile, those data could provide useful information for developing new therapeutic strategy for DR.

Keywords: Muller cells • diabetic retinopathy