Protective effect of miR-30b on retinal ganglion cells against oxygen-glucose deprivation in vitro

Authors:Huang Chanjuan,  Huo Yan,  Chen Chen,  Ai Liqianyu,  Zhou Yuanguo,  Ye Jian
DOI: 10.3760/cma.j.issn.2095-0160.2016.05.004
Published 2016-05-10
Cite as Chin J Exp Ophthalmol, 2016,34(5): 396-401.

Abstract

Background

Retinal ganglion cell (RGCs) death following ischaemic insult is the major cause of a number of vision-threatening diseases.Recent studies confirmed that micro RNA (miR-30b) can alleviate hypoxy-induced cardiac injury.However, whether miR-30b can protect RGCs against oxygen-glucose deprivation damage is still not ellucidated.

Objective

The aim of this study was to investigate the protective effect of miR-30b on RGCs damage caused by oxygen-glucose deprivation.

Methods

The retinas were isolated from the eyeballs of eight SD rats aged postnatal 24 hours and RGCs were primarily cultured.The cells were divided into the recombinant adeno-associated virus (rAVV) control group, rAAV-miR-30b mimic group and AAV-miR-30b inhibitor group.Then the cells were transfected using rAVV-miR plasmid, rAAV-miR-30b mimic plasmid and AAV-miR-30b inhibitor plasmid, respectively for 6 days with the RGCs∶AAV as 1∶10 000.The cells were cultured with low glucose medium in hypoxygen incubator (5%CO2, 17%N2, 3%O2) or 5%CO2incubator respectively for 24 hours.Cell viability was detected by cell counting kit-8 assay.The expression of Tubulin Ⅲ, a neuron specific marker, was detected by immunofluorescence technology to evaluate the survival of RGCs.The apoptosis and necrosis of the cells were assessed by Hoechst/PI double staining.

Results

The RGCs grew well with round shape and 1—3 processes 7 days after cultured in the normal cells.However, the RGCs were diminished and the cell process disrupted in the oxygen-glucose deprivation group.The relative vability of the cells was 3.310±0.162 in the rAAV-miR-30b mimic group, which was significantly higher than 0.949±0.141 in the rAAV-miR-30b inhibitor group and 0.900±0.181 in the rAAV-miR control group(t=10.508, 10.296, both at P<0.001). It was positively expressed in survival RGCs, with the red fluorescence.The number of Tubulin Ⅲ+cells was (13.800±1.924)/field in the rAAV-miR-30b mimic group, showing a significant increase in comparison with (0.600±0.548)/field in the rAAV-miR-30b inhibitor group and (0.800±1.304)/field in the rAAV-miR control group (t=15.141, 14.912, both at P<0.001). Significant differences were found in the apoptosis rate and necrosis rate among the rAAV-miR-30b mimic group, rAAV-miR control group and PBS group (F=10.851, P=0.002; F=6.378, P=0.013), and the apoptosis rate and necrosis rate in the rAAV-miR-30b mimic group were considerably lower than those in the rAAV-miR control group and PBS group (all at P<0.05).

Conclusions

The oxygen-glucose deprivation models can be established in RGCs by hypooxygic and low-glucose cultivation.rAAV encoding miR-30b mimics transfection can protect RGCs against oxygen-glucose deprivation damage.

Key words:

MicroRNAs/metabolism; Retinal ganglion cells; Cell hypoxia/physiology; Glucose/physiology; RNA interference; Cytoprotection/drug effects; Rats, Sprague-Dawley; Oxygen-glucose deprivation

Contributor Information

Huang Chanjuan
Department of Ophthalmology, Daping Hospital, Third Military Medical University, Chongqing 400042, China
Huo Yan
Chen Chen
Ai Liqianyu
Zhou Yuanguo
Ye Jian
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Updated: October 9, 2019 — 2:07 am