| Background:The optic nerve(ON) is part of the central nerve system(CNS). The axons of retinal ganglion cells(RGCs) comprise the ON. The state of RGCs partly reflects the function of ON.The regeneration and functional recovery of a damaged ON remain challenging due to many regulatory factors. Some of the most significant problems associated with CNS regeneration include glial scar formation, neurotrophic factor deficits, and the presence of inhibitory proteins. Among them, the inhibitory influences are the most impo rtant contributing factors. Semaphorin3A(Sema3A) is a very potent repulsive axon guidance cue and an important inhibitory factor involved in CNS repair following damage. The binding of Sema3 A to Neuropilin1(NRP1) induces a structural alteration in Plexin A1-A4(Plex A1-A4) leading to the release of PlexA1-A4 from a self-inhibited status and the initiation of downstream signaling, which in turn induces growth cone collapse and inhibits axon elongation.The p38 MAPK signaling pathway is involved in apoptosis in a variety of cell types. Sema3 A activates p38 MAPK phosphorylation and promotes neural progenitor cell apoptosis. Furthermore, Sema3 A can reduce myelin lipid phagocytosis by macrophages, which in turn inhibits myelin regeneration of the CNS and blocks neu rotrophic factor transport and recovery of the damaged nerves.MicroRNAs(mi RNAs) are endogenous small molecules that reduce the expression levels of proteins by binding to the three prime untranslatedregion(3’UTR) of the targeted mRNAs and inhibiting or degrading the mRNAs, allowing them to regulate cell proliferation, differentiation, and apoptosis. Many mi RNAs are expressed specifically in the mammalian brain and retina and are dynamically regulated during development, suggesting that this group of mi RNAs may be associated with neural development. Further investigation into Sema3 A and retina-associated mi RNAs may provide a new window of opportunity for exploring the treatment of ON damage via Sema3 A inhibition.Objective:This study is to reveal the expression and effect of Sema3 A and its signal channel in retina after ON damage. It will confirm microRNA-30b(mi R-30b) as a candidate modulator of Sema3 A expression and investigate the molecular mechanism ofmi R-30 b protects RGCs. As a result, it will provide a new strategy for the clinical treatment of ON injury.Methods:1.The expression patterns of mi R-30 b, Sema3 A, NRP1, PlexA1, p-p38 MAPK, and active caspase-3 in the retinas from normal rats and rats with a damaged ONwere investigated by using q RT-PCR and Western blot methods. The locations of Sema3 A in the retinas from normal rats and rats with a damaged ON were investigated by using immunohistochemistry.2. mi R-30 b bound tothe three prime untranslated region(3’UTR) of Sema3 Aby using a dual-luciferase reporter assay.In vitrocultures of rat RGCs were transfected with r AAV-miR-30 b mimic, rAAV-miR-30 b inhibitor, r AAV-mi RNA NC, or PBS at 24 h after plating. Protein expression levels of Sema3 A, NRP1, Plex A1, p-p38 MAPK, and active caspase-3 were measured at 7 d after the initial plating by Western blot method.Immediately after the ON of rat was damaged, r AAV-mi R-30 b mimic, r AAV-miR-30 b inhibitor, r AAV-miRNA NC, or PBS was injected into the vitreous. The expression levels of mi R-30 b and Sema3 A were analyzed by using q RT-PCR and Western blot methods at 3 d and 7 d post-injury.3. Rat RGCs were transfected with one of three different si RNA duplexes targeting Sema3 Aor with siRNA-NC at 24 hours after plating. Sema3 A protein expression levels were determined at 5 d post-plating by Western blot method.si RNA2 having the most efficient knockdown of Sema3 A expression in the three si RNA was selected for use in subsequent experiments. Rat RGCs were transfected with si RNA2-Sema3 A at 24 h after plating, followed by transfection with the rAAV-mi R-30 b mimic or rAAV-mi R-30 b inhibitor at 5 d post-plating. Protein expression levels of Sema3 A, NRP1 and p-p38 MAPK were determined at 9 d post-plating by Western blot method.4. In vitrocultures of rat RGCs were transfected with r AAV-miR-30 b mimic, r AAV-miR-30 b inhibitor, rAAV-mi RNA NC, or PBS at 24 h after plating. Rates of apoptosis were determined by flow cytometry at 5 d post-plating. The RGCs were stained with anti-GAP-43 antibodies and axon lengths were analyzed at 7 d post-plating.5. Immediately after the ON was damaged, rAAV-mi R-30 b mimic, rAAV-miR-30 b inhibitor, rAAV-miRNA NC, or PBS was injected into the vitreous.The survival rates of RGCs were investigated by fluorogold retrogradely labeled. The recovery of optic nerve function was investigated by FVEP.Results:1. After the rat ON was damaged with an arterial clamp, mi R-30 b expression levels in the retina increased and peaked at 3 d relative to mi R-30 b expression levels in control retinas. Sema3 A mRNA expression levels peaked at 7 d post-injury. Sema3 A protein expression levels increased significantly in retinas associated with ON injury relative to control retinas, reaching a peak value at 7 d. The protein expression levels of NRP1, PlexA1, p-p38 MAPK, and active caspase-3 also increased significantly in retinas associated with ON injury relative to control retinas. In the undamaged retina, Sema3 A protein expression was detected in the inner nuclear layer(INL) and the ganglion cell layer(GCL). Sema3 A expression levels in the INL and GCL increased significantly at 7 d in retinas associated with ON injury compared to control retinas.2. Results of the dual-luciferase reporter assay showed that the relative luciferase activity was significantly lower in the treatment group relative to the control or mutation groups.mi R-30 b bound to Sema3 A at the sequence UGUUUACA. In vitrocultures of rat RGCs were transfected with r AAV-mi R-30 b mimic, rAAV-mi R-30 b inhibitor, r AAV-miRNA NC, or PBS at 24 h after plating. The changes in NRP1, PlexA1, p-p38 MAPK, and active caspase-3 protein expression levels were similar to those of Sema3 A, i.e., lower protein expression levels were detected in the mi R-30 b mimic group relative to the remaining three groups, and higher protein expression levels were detected i n the mi R-30 b inhibitor group relative to the remaining three groups. Following ON damage, r AAV-miR-30 b mimic, r AAV-mi R-30 b inhibitor, r AAV-mi RNA NC, or PBS was injected into the vitreous. The expression levels of mi R-30 b and Sema3 A were analyzed at 3 d and 7 d post-injury. mi R-30 b mRNA expression levels were higher in the mi R-30 b mimic group and lower in the mi R-30 b inhibitor group than in the other three groups. In contrast, the mRNA and protein expressions of Sema3 A were the lowest in the mi R-30 b mimic group and the highest in the mi R-30 b inhibitor group.3. The RGCs transfected with si RNA2-Sema3 A exhibited the most efficient knockdown of Sema3 A expression. Rat RGCs were transfected with siRNA2-Sema3 A at 24 h after plating. Subsequently, the RGCs were transfected with rAAV-miR-30 b mimic or r AAV-miR-30 b inhibitor at 5 d after plating. Total proteins were extracted at 9 d after plating, and the expression levels of Sema3 A, NRP1 and p-p38 MAPKwere not significantly different between the r AAV-mi R-30 b mimic and the rAAV-mi R-30 b inhibitorgroups.4. In vitrocultures of rat RGCs were transfected with r AAV-miR-30 b mimic, r AAV-miR-30 b inhibitor, r AAV-mi RNA NC, or PBS at 24 h after plating. The rate of apoptosis was the lowest in the mi R-30 b mimic group and highest in the miR-30 b inhibitor group. RGC axon length was longest in the miR-30 b mimic group and shortest in the mi R-30 b inhibitor group at 7 dafter plating.5. Following ON damage, r AAV-mi R-30 b mimic, r AAV-mi R-30 b inhibitor, r AAV-miRNA NC, or PBS was injected into the vitreous. The survival rate of RGCs was the highest in the mi R-30 b mimic group and lowest in the mi R-30 b inhibitor group. The N1-P1 wave amplitude was the widest in the miR-30 b mimic group and thenarrowest in the mi R-30 b inhibitor group.Conclusions:1. Sema3 A is the target gene of mi R-30 b in vitro and in vivo.2. mi R-30 b inhibited Sema3 A expression in RGCs, decreased RGC apoptosis and increased the survival rate of RGC after optic nerve damaged by inhibiting p-p38 MAPK and active caspase-3.3. mi R-30 b inhibited Sema3 A expression in RGCs, promoted axon outgrowth and recovery of optic nerve functionafter damaged by reducing the binding capability of Sema3 A to NRP1/Plex A1. |
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