Saxena, Kartik
Description
Inflammation is established as a key factor in mediating the
progression of a number of retinal degenerations, including both
wet and dry age-related macular degeneration (AMD) [1-3].
MicroRNAs (miRNAs) are a class of endogenously occurring
non-coding RNA (ncRNA) molecules that are gaining momentum as
therapeutic targets for treating a number of human conditions
[4-6] and have been identified to modulate inflammation [7]. The
purpose of this study is to...[Show more] investigate the modulation of miRNAs
in a model of retinal degeneration, the light damage model. MiRNA
and their potential roles in mitigating retinal inflammation will
also be investigated in animals treated with 670nm red light
therapy.
Albino rats raised in dim cyclic light conditions (5lux; 12hr on,
12hr off; controls) were exposed to bright continuous light
(1000lux) for 24 hours and returned to dim light conditions for
0, 3 or 7 days. At each timepoint animals were culled and their
eyes removed and processed either for histological analyses or
RNA based analyses. For histology eyes were fixed in 4%
paraformaldehyde, cryoprotected and sectioned to determine the
photoreceptor cell death using TUNEL or perform
immunohistochemistry experiments using Vimentin and IBA1 or in
situ hybridisation for Ccl2 and miR-124-3p. RNA was extracted
from dissected retinas, reverse transcribed and used for low
density array and qPCR analysis to determine the expression
changes of genes and miRNAs. Immortalised cell lines were also
used for performing cell transfections and similar RNA based
analyses as above.
Intense light exposure for 24 hours led to differential
expression of a number of miRNAs, 37 of which were significantly
modulated by 2-fold or more. Of those, 19 may potentially
regulate the inflammatory immune response observed in the model.
MicroRNAs -125-3p, -155, -207, -347, -449a, -351, and -542-3p are
all upregulated at 24 hours of exposure along with peak
photoreceptor cell death. The miRNAs -542-3p and -351 reached
maximum expression at 7 days after exposure, while -125-3p, -155,
-207, -347, and -449 reached a peak expression at 3 days.
MiR-124-3p expression showed significant downregulation post
intense (1000lux) light induced damage to the rat retina. Its
expression was localized predominantly to the Müller glia and
showed co-localisation with expression of a target gene, Ccl2
(which is a potent chemoattractant molecule responsible for
targeted monocyte recruitment in the retina). Luciferase assays
in MIO-M1 and HeLa cells confirmed direct binding between
miR-124-3p and the CCL2-3’UTR. Additionally, in vitro
overexpression of miR-124-3p in Müller cells using miRNA mimics
significantly inhibited CCL2 upregulation post stimulation with
inflammatory cues. Post mortem human tissue showed a similar
expression profile for miR-124-3p in the retina. Changes is miRNA
expression were also seen in 670nm light treated retinas with or
without bright light induced damage, along with the modulation of
chemokine gene expression in the pre-treated retinas.
Differential expression of miR-351 and miR-155 was confirmed
using qPCR, both of which are predicted to target inflammation
related genes.
The data indicate that miRNAs are involved in modulating the
inflammatory immune response elicited during retinal
degeneration. Indeed we found a number of potential candidates,
including miR-124-3p, which could prove to be novel therapeutic
interventions in mitigating retinal inflammation and the
consequent photoreceptor death. 670nm light therapy mitigated
retinal inflammation including the modulation of specific miRNAs
in the retina.
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