Protective effect of miR-30b on retinal ganglion cells against oxygen-glucose deprivation in vitro / 中华实验眼科杂志

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％ CO2 incubator 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.