Abstract
Purpose: :
To examine the inhibitory role of tissue inhibitor of metalloproteinases 3 (timp3) on angiogenesis in diabetic retinas, and to study this regulatory mechanism by microRNA (miRNA) at post-transcriptional level.
Methods: :
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 timp3 was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blot. Bioinformatics analysis was used to predict miRNAs that target timp3, and luciferase assay in vitro was performed to confirm miRNA and 3’UTR of timp3 interaction. The level of miRNA of interest in diabetic retina was detected by qRT-PCR. Further glyoxal (AGEs stress) was used to treat rat Müller cells (rMC-1 cell line) in vitro, and then the mRNA level of timp3 and mir365 were detected.
Results: :
In diabetic retina, timp3 expression level showed significant decreased from 4 week diabetes onset as compared to normal controls. Using Targetscan (5.1 version) software, 3’UTR of timp3 mRNA was predicted to be the target of mir365. Their interaction was confirmed by in vitro double luciferase activity assay in HEK293 cell. In diabetic retina, the levels of mir365 and mir221 (the known miRNA to target timp3) increased in 1 week and 2 week diabetes, but no significant change was observed in 4 week and 6 week diabetes. In rMC-1, the expression of mir365 was enhanced and timp3 was decreased after 2mM glyoxal treatment.
Conclusions: :
In diabetic retinas, timp3 expression level significantly down-regulated from 4 weeks after diabetes onset. Since vascular endothelial factor -A (VEGF-A) is increased in diabetic retinas, the decreased expression of timp3 that is known angiogenesis inhibitor may contribute or even aggravate the angiogenic potential in diabetic retina. Our data also demonstrated that in post-transcriptional level, mir365 is a new miRNA to target timp3, and it has synegic effects with mir221 to regulate timp3 expression in a DR model. The increased level of mir365 and mir221 occurred in 1 week and 2 week after diabetes onset, which proceded the change of timp3 expression. These findings implicate the anti-angiogenic potential of mir365 through the regulatory effect on timp3 in diabetic retinas.
Keywords: diabetic retinopathy • neovascularization • retina