MiR-30b Regulates Polarity And Protects Retinal Ganglion Cells Against Oxygen-glucose Deprivation In Vitro

Background:Optic nerve belongs to the central nervous system,and its ability to regenerate after injury is extremely limited.Optic nerve consists of axons of retinal ganglion cells.Therefore one of the important factors that affect regeneration is axon growth after the optic nerve injury.Recent studies have found that the polarity determines the direction of synaptic growth,contributes to functional recovery.And direction of the synaptic growth plays an important role in regeneration of optic nergve after injury.RGCs are typical consisting of a long axon and multiple branching dendrites,lies at the core of the principle of dynamic polarization,whereby information flows from dendrites toward the soma and to the axon.Micro-RNAs contribute essential roles in controlling neuronal polarity.Our previous studies have found that the expression of miR-30 b upregulated dramatically after optic nerve injury,nonetheless overexpression of miR-30 b inhibits Sema3 A expression in vitro.Sema3 A is a neuronal guidance molecule.Recent studies have found that Sema3 A contribute to hippocampal neuronal polarity by regulating the cGMP/cAMP.But there is no evidence indicated whether miR-30 b would affect RGCs polarity through regulating Sema3 A expression.In addition,secondary injury after optic nerve injury can cause irreversible damage to RGCs,oxygen-glucose deprivation damage play an important role in it.Recent studies confirmed that micro-RNA(miR-30b)can alleviate hypoxy-induced cardiac injury.However,whether miR-30 b can protect RGCs against oxygen-glucose deprivation damage is still not ellucidated.In this study,we used recombinant adeno-associated virus to improve transfection miR-30 b content in RGCs,and observed the effect of miR-30 b on RGCs' double polarity by suppression of Sema3 A.What is more,miR-30 b of RGCs plays a protective role in oxygen-glucose deprivation.Methods:1.The primary RGCs were transfected with rAVV-miR virus,rAAV-miR-30 b mimic virus and rAAV-miR-30 b inhibitor virus,respectively for 7 days.The expression of Tubulin ?,a neuron specific marker,was detected by immunofluorescence to label the neurite of RGCs and MAP2 to evaluate the dendritic of RGCs.the use of IPP image analysis length of axon/dendrite.2.The primary RGCs were transfected with rAVV-miR virus,rAAV-miR-30 b mimic virus,rAAV-miR-30 b inhibitor virus and SiRNA-Sema3 A group,PBS group.Using Western blotting methods detect Sema3 A expression in these groups.And Protein expression levels of PKA,CRMP-2 were determined at 7 d post-plating by Western blot in the high Sema3 A group,rAAV-miR-30 b mimic group,rAVV-miR group and the control group.3.The cells were transfected with rAVV-miR virus,rAAV-miR-30 b mimic virus and rAAV-miR-30 b inhibitor virus,respectively for 6 days.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 ?,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 and concl usions:1.In vitro overexpression of miR-30 b in primary culturned RGCs result in significantly increase of axon length,and significantly decrease of the number of dendritic.What is more,overexpression of miR-30 b increased the rate of bipolar RGCs.2.In vitro overexpression of miR-30 b in primary culturned RGCs result in significantly decrease expression of Sema3 A.The sema3 A protein in the siRNA-Sema3 A group was obviously lower.Overexpression of miR-30 b obviously increase the number of p-PKA opposite decrease the the number of CRMP-2(T514).On the contrary,The p-PKA is lower,and the CRMP-2(T514)is increase in the high concentration of Sema3 A group.3.The RGCs were diminished and the cell process disrupted in the oxygen-glucose deprivation group.Overexpression of miR-30 b obviously increased the number of Tubulin ? positive cells and decreased the rate of apoptosis and necrosis in the oxygen-glucose deprivation group.My study results suggest overexpression of miR-30 b in primary culturned RGCs can promote the growth of axons,and inhibit the growth of dendrites,and help maintain RGCs dual polarity.The effects are mediated by suppression Sema3 A expression.Besides,overexpression of miR-30 b can protect RGCs against oxygen-glucose deprivation damage.