Background/Aims: Retinal Müller cells could be induced to differentiate into retinal ganglion cells (RGCs), but RGCs derived from Müller cells have defects in axon growth, leading to a defect in signal conduction. In this study we aimed to explore the role of miR-124 in axon growth of RGCs derived from Müller cells. Methods: Müller cells were isolated from rat retina and induced to dedifferentiate into retinal stem cells. The stem cells were infected by PGC-FU-Atoh7-GFP lentivirus and then transfected with miR-124 or anti-miR-124, and the length of axon was compared. Furthermore, the cells were injected into the eyes of rat chronic ocular hypertension glaucoma model and axon growth in vivo was examined. The targeting of CoREST by miR-124 was detected by luciferase assay. Results: In retinal stem cells, the length of axon was 1,792±64.54 µm in miR-124 group, 509±21.35 µm in control group, and only 87.9±9.24 µm in anti-miR-124 group. In rat model, miR-124 promoted axon growth of RGCs differentiated from retinal stem cells. Furthermore, we found that miR-124 negatively regulated CoREST via directly targeting the binding site in CoREST 3′ UTR. Conclusions: We provide the first evidence that miR-124 regulates axon growth of RGCs derived from Müller cells, and miR-124 has translational potential for gene therapy of glaucoma.

Glaucoma is a serious eye disease of severe loss in visual function due to selective and progressive death of retinal ganglion cells [1, 2]. Recent studies suggest that retinal Müller cells could be induced to dedifferentiate into potential retinal stem cells [3-5]. The dedifferentiated retinal Müller cells then differentiate into different types of neurons such as retinal ganglion cells (RGCs). Our previous studies showed that RGCs derived from Müller cells had defects in axon growth, leading to a defect in signal conduction [6, 7].

Semaphorin is a group of secretory membrane associated glycoprotein. Sema3A is a member of semaphorin family and plays an important role in navigation and growth of nerve axons [8, 9]. Neuropilin-1 (NRP-1) is a specific receptor of Sema3A with a high affinity and interacts with PlexinA, a receptor in growth cone of RGCs. When Sema3A binds to NRP-1/PlexinA receptor complex, signaling cascade is activated including G protein, Racl, and CRMP, leading to axon growth [10, 11]. Thus we speculate that the lack of NRP-1 or/and Sema3A in the axons of RGCs differentiated from stem cells may contribute to the defects in axon growth.

miRNAs are small noncoding RNAs that recognize the 3′ untranslated region (UTR) of target genes and repress gene expression [12, 13]. Role of miRNAs in the regulation of gene expression in RGCs was investigated previously [14]. In addition, miR-124 regulated the expression of NRP-1 in growth cone of RGCs, and promoted the binding of Sema3A with NRP-1 [14, 15]. Therefore, this study aimed to explore the potential role of miR-124 in axon growth of ganglion cells derived from Müller cells both in vitro and in vivo.

Dedifferentiation of purified Müller cells into retinal stem cells and ganglion cells

The enrichment of Müller cells and differentiation into RGCs were performed as previously described [7].

Luciferase assay

Rat RGC-5 cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C with 5% CO2. CoREST 3′ UTR–luc plasmid, pCMV-β-gal, pLKD.CMV.GFP.U6, or pLKD.CMV.GFP.U6-miR-124, pLKD.CMV.G&PR, pLKD.CMV.G&PR. U6-anti-miR-124 were co-transfected into RGC-5 cells, and 48 hours later cell lysates were assayed for luciferase activity in triplicate.

Lentivirus vectors and intraocular injection

Lentivirus vectors pLKD.CMV.GFP.U6-miR-124 and pLKD.CMV.G&PR.U6-anti-miR-124 were constructed by Auragene Bioscience (Changsha, China). The neurosphere cells were infected by PGC-FU-Atoh7-GFP lentivirus. After 48 h, the neurospheres were divided into five groups. Group A: without transfection; Group B: transfected by empty vector pLKD.CMV.GFP.U6; Group C: transfected by empty vector pLKD.CMV.G&PR. U6; Group D: transfected with pLKD.CMV.G&PR.U6-anti-miR-124; Group E: transfected by pLKD.CMV.GFP. U6-miR-124. The neurospheres of each group were dissociated into single stem cells with Accutase and collected at a concentration of 1x104 cells/ml. The stem cells were cultured in 1 ml differentiation medium supplemented with brain-derived neurotrophic factor (BDNF) (1 ng/ml) (Peprotech), retinoic acid (1 µM) (Sigma) and 1% FBS. The anterograde tracer cholera toxin b subunit conjugated to alexa-594 (CTb-594, Molecular Probes, Zug, Switzerland) was added to each well. At day 14, the cells were stained with RGC markers Thy1.1 and Brn-3b to calculate the length of axon. In vivo, the rats were anesthetized by i.p. injection of pentobarbital. 5 µl of retinal stem cells from different groups (1x104 cells/mL) were injected using a 33-gauge needle to insert tangentially through the sclera into subretinal space. 5 µL BDNF (1 ng/mL) (Peprotech) and 100 ng RA (1 µM) (Sigma) were injected together to maintain the growth of the cells. At day 14, the eyes were dissected for further analysis.

Immunocytochemical and Edu labeling analysis

Immunocytochemical and Edu labeling analysis were performed as previously described [6] with primary antibodies listed in Table 1 and secondary antibodies conjugated with FITC or TRITC (Sigma). The neurospheres were incubated with Edu (1: 1, 000 dilution, Riboio, Guangzhou, China) at 37°C overnight. The cells were washed and fixed in 4% paraformaldehyde for 30 min, then incubated with Apollo buffer (RiboBio) at room temperature for 30 min in the dark. The cells were washed with 0.5% Trixton X-100 and stained with DAPI (Beyotime, Wuhan, China) and images were captured using fluorescent microscopy (Leica, Solms, Germany).

Table 1.

List of antibodies used in this study

List of antibodies used in this study
List of antibodies used in this study

RT-PCR analysis

Total RNA was isolated from cells using Trizol reagent and reverse transcribed to cDNA. PCR was performed using cDNA, SYBR Green mix and the primers listed in Table 2. Amplification conditions were: 15 sec at 95°C (one cycle); 5 sec at 95°C, 5 sec at annealing temperature and 30 sec at 72°C (45 cycles). The data were analyzed by ABIViia7 (ABI, Foster City, CA, USA).

Table 2.

List of primers used in this study

List of primers used in this study
List of primers used in this study

Western blot analysis

Cells were lysed in RIPA buffer (RiboBio, Guangzhou, China) containing protease and phosphatase inhibitors. Lysates were separated on SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk for 1h, then incubated with primary antibodies for 1 h at room temperature. The membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 h, then visualized with enhanced chemiluminescence reagent.

Establishment of chronic ocular hypertension glaucoma model

Animals were used following the Guidelines for Animal Experiments of Central South University (Changsha, China). All animal experiments were conducted with the approval of Animal Research Committee, Xiangya School of Medicine, Central South University (Changsha, China). Ocular hypertension was induced using a previously described method [16]. The rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (0.4 ml/100 g; Sigma, St. Louis, MO, USA) and placed in front of a slit lamp equipped with a 532 nm diodelaser (Carl Zeiss, Germany). 1% proparacaine (AlconPharm Inc., TX, USA) was applied to the right eye (experimental eye) as a topical anesthetic before laser photocoagulation. Then 50-60 laser pulses were directed to the trabecular meshwork 270°around the circumference of the aqueous outflow area and 15-20 laser spots on each episcleral aqueous humor drainage vein of the right eye. The left eye without any treatment served as control. Intraocular pressure (IOP) was measured bilaterally using a digital tonometer (Tonopen XL, Reichert, USA) on day 3, day 7, day 14, day 28, day 60 after laser photocoagulation.

TUNEL assay

The apoptosis of RGCs was quantified by TUNEL staining using DeadEndTM Fluorometric TUNEL System (Beyotime Institute of Biotechnology, Wuhan, China). Frozen tissue sections were fixed with formaldehyde, then rinsed in PBS and treated with 1% Triton X-100 in PBS for 2 min on ice. Slides were incubated in recombinant terminal deoxynucleotidyl transferase (rTdT) incubation buffer for 1 h at 37°C. The negative control sections were incubated with the buffer without rTdT. Apoptotic cells were counted from three randomly selected sections in the GCL layer (from three rats and from six microscopic fields in each section including two optic disc areas, two peripapillary areas, and two peripheral areas). The slides were observed under confocal microscope with the observer blind to the grouping of the slides.

Axonal growth analysis

The axon growth was examined 2 weeks after intraocular injection of retinal stem cells from different groups. The rats were intracardially perfused with 4% paraformaldehyde (PFA) and the retina were rapidly dissected. Next, the retina were washed and dehydrated in increasing concentrations of ethanol (50, 80, and 96%) at room temperature for 1 h and kept in 100% ethanol overnight. The clearing solution composed of the mixture of benzyl alcohol and benzylbenzoate (1: 2) (Sigma-Aldrich) was then added. The whole retina were mounted and images were captured using a confocal Leica SP5 inverted microscope (Leica, Mannheim, Germany).

Statistical analysis

Data were expressed as mean ± SD and analyzed using SPSS 13.0 (Chicago, IL, USA). Comparison was performed with one-way ANOVA or Student’s t-test. P <0.05 was considered significant.

Müller cells are induced to dedifferentiate into retinal stem cells

Müller cells were isolated and purified, then cultured in stem cell conditioned medium, and the single cells gradually aggregated. The proliferation was clonal, and a few spherical cell spheres appeared. The cells within the spheres were positive for retinal stem cell markers Nestin and Ki-67 (Fig. 1A). After seven days of culture, the spheres became further rounded and bigger, resembling neurospheres (Fig. 1B). After ten days, the spheres showed significant increase in number and size, were stained positive for Nestin and Ki-67 (Fig. 1C), and the nuclei were stained red by EdU staining (Fig. 1D). These data indicate that retinal Müller cells dedifferentiate into retinal stem cells and keep the capacity of proliferation.

Fig. 1.

Characterization of stem cells derived from Müller cells. Müller cells were purified and cultured in stem cell conditioned medium for 3, 7 and 10 days, and they formed neurospheres. Stem cells within the spheres showed positive staining of retinal stem cell-specific markers Nestin and Ki-67 (A, B, C). Edu staining showed that cell spheres had the capacity of effective proliferation (D). Scale bar: 100 µm. RT-PCR and Western blot analysis of the expression of Nestin and Ki-67 (E, F). Lane M: DNA marker.

Fig. 1.

Characterization of stem cells derived from Müller cells. Müller cells were purified and cultured in stem cell conditioned medium for 3, 7 and 10 days, and they formed neurospheres. Stem cells within the spheres showed positive staining of retinal stem cell-specific markers Nestin and Ki-67 (A, B, C). Edu staining showed that cell spheres had the capacity of effective proliferation (D). Scale bar: 100 µm. RT-PCR and Western blot analysis of the expression of Nestin and Ki-67 (E, F). Lane M: DNA marker.

Close modal

By RT-PCR and Western blot analysis, we found that the cells in the spheres expressed stem cell markers Nestin and Ki67, which were not expressed in Müller cells. The control group showed no expression of Nestin or Ki67 (Fig. 1E, F).

Axon growth of RGCs is correlated with reduced level of miR-124

Retinal stem cells derived from Müller cells were infected by PGC-FU-Atoh7-GFP lentivirus to promote their differentiation into RGCs. Immunofluorescence analysis was performed to detect Thy1.1 and Brn-3b, and the length of RGCs axons was calculated. Few cells were positive for Thy1.1 and Brn-3b (Fig. 2A). After 3 days, stem cells began to differentiate into RGCs. Some cells turned into spindle-shaped, and showed strong staining of Thy1.1 and Brn-3b (Fig. 2B). At 7 days, the length of axons increased and grew towards two ends (Fig. 2C). At 14 days, axons of RGCs almost stopped growing (Fig. 2D).

Fig. 2.

Characterization of RGCs derived from Müller cells. The axons of RGCs grew gradually under differentiation medium (A, B, C). After 14 days, axons almost stopped growing (C, D). The length of axons was calculated. *P<0.05, **P<0.01 vs. 0 day. RT-PCR and Western blot analysis showed that Sema3A and NRP-1 at both mRNA and protein levels reduced on 3 days, 7 days, 14 days and 28 days after infection by PGC-FU-Atoh7-GFP lentivirus, but the expression of CoREST and REST increased (E, F). RT-PCR analysis demonstrated that the expression of miR-124 gradually decreased. *P<0.05 vs. 0 day (G).

Fig. 2.

Characterization of RGCs derived from Müller cells. The axons of RGCs grew gradually under differentiation medium (A, B, C). After 14 days, axons almost stopped growing (C, D). The length of axons was calculated. *P<0.05, **P<0.01 vs. 0 day. RT-PCR and Western blot analysis showed that Sema3A and NRP-1 at both mRNA and protein levels reduced on 3 days, 7 days, 14 days and 28 days after infection by PGC-FU-Atoh7-GFP lentivirus, but the expression of CoREST and REST increased (E, F). RT-PCR analysis demonstrated that the expression of miR-124 gradually decreased. *P<0.05 vs. 0 day (G).

Close modal

RT-PCR and Western blot analysis showed that both mRNA and protein levels of Sema3A and NRP-1 were reduced but the expression of CoREST and REST increased on 3 days, 7 days, 14 days and 28 days after the cells were infected by PGC-FU-Atoh7-GFP lentivirus (Fig. 2E, F). Furthermore, the expression of miR-124 decreased gradually (Fig. 2G). These data suggest that in vitro axon growth of RGCs differentiated from retinal stem cells is limited, along with reduced expression of Sema3A and NRP-1 and reduced miR-124 level.

miR-124 promotes axon growth of RGCs differentiated from retinal stem cells in vitro

Next we transfected Müller cells derived RGCs with pLKD.CMV.GFP.U6-miR-124, pLKD. CMV.G&PR.U6-anti-miR-124, pLKD.CMV.GFP.U6 and pLKD.CMV.G&PR.U6 to explore the effect of miR-124 on axon growth of RGCs. After 14 days, immunofluorescence analysis was performed to detect Thy1.1and Brn-3b. The length of axon of RGCs was the shortest in anti-miR-124 group (87.9±9.24 µm, n=5, Fig. 3B) and the longest in miR-124 group (1, 792±64.54 µm, n=5, Fig. 3E), and was 509±21.35 µm, 497±18.25 µm and 501±19.32 µm in control group, con-anti-miR-124 group and con-miR-124 group, respectively (n=5, Fig. 3A, C, D).

Fig. 3.

Characterization of RGCs with overexpression or knockdown of miR-124. After retinal stem cells derived from Müller cells were infected by PGC-FU-Atoh7-GFP lentivirus, the cells were transfected with pLKD.CMV.G&PR.U6-anti-miR-124 (anti-miR-124 group), pLKD.CMV.G&PR.U6 (Con-anti-miR-124 group), pLKD.CMV.GFP.U6 (Con-miR-124 group) and pLKD.CMV.GFP.U6-miR-124 (miR-124 group), then cultured in DMEM/F12 differentiation medium. Double immunocytochemical analysis showed that RGCs expressed both Thy1.1 and Brn-3b. Scale bar: 100 µm. A. control group: cells infected by PGC-FU-Atoh7-GFP lentivirus only. B. anti-miR-124 group. C. con-anti-miR-124 group. D. con-miR-124 group. E. miR-124 group. Immunocytochemical analysis showed that the axon of miR-124 group was the longest. **P<0.01 vs. Control group. F, G. RT-PCR and Western blot analysis of Sema3A, NRP-1, CoREST and REST expression in each group. H. RT-PCR analysis of the expression of miR-124 in each group. **P<0.01 vs. Control group.

Fig. 3.

Characterization of RGCs with overexpression or knockdown of miR-124. After retinal stem cells derived from Müller cells were infected by PGC-FU-Atoh7-GFP lentivirus, the cells were transfected with pLKD.CMV.G&PR.U6-anti-miR-124 (anti-miR-124 group), pLKD.CMV.G&PR.U6 (Con-anti-miR-124 group), pLKD.CMV.GFP.U6 (Con-miR-124 group) and pLKD.CMV.GFP.U6-miR-124 (miR-124 group), then cultured in DMEM/F12 differentiation medium. Double immunocytochemical analysis showed that RGCs expressed both Thy1.1 and Brn-3b. Scale bar: 100 µm. A. control group: cells infected by PGC-FU-Atoh7-GFP lentivirus only. B. anti-miR-124 group. C. con-anti-miR-124 group. D. con-miR-124 group. E. miR-124 group. Immunocytochemical analysis showed that the axon of miR-124 group was the longest. **P<0.01 vs. Control group. F, G. RT-PCR and Western blot analysis of Sema3A, NRP-1, CoREST and REST expression in each group. H. RT-PCR analysis of the expression of miR-124 in each group. **P<0.01 vs. Control group.

Close modal

By RT-PCR and Western blot analysis we found that the expression levels of Sema3A and NRP-1 were upregulated in miR-124 group but downregulated in anti-miR-124 group (Fig. 3F, G). Meanwhile, the expression of miR-124 was significantly upregulated in miR-124 group (Fig. 3H). These results indicate that miR-124 promotes axon growth of RGCs differentiated from retinal stem cells in vitro.

miR-124 targets CoREST 3′UTR to suppress CoREST expression

Bioinformatics analysis showed that 3′UTR of CoREST has a predicted binding site for miR-124 (Fig. 4A). Luciferase assays with the CoREST 3′ UTR luciferase reporter revealed that miR-124 targeted CoREST 3′UTR to suppress CoREST expression. miR-124 expression vector (U6-miR-124) repressed luciferase activity from CoREST 3′ UTR-luc (49.6% of control, P< 0.05), while anti-miR-124 expression vector (U6-anti-miR-124) stimulated luciferase activity (3.1 fold compared to control, P< 0.01) (n=3, Fig. 4B). Taken together, these results demonstrate that miR-124 negatively regulates CoREST via targeting the binding site in CoREST 3′ UTR.

Fig. 4.

miR-124 targets CoREST 3′UTR to suppress its expression. Bioinformatics analysis of miR-124 binding site in 3′UTR of CoREST (A). Luciferase assays with the CoREST 3′ UTR luciferase reporter in RGC-5 cells (B). RGC-5 cells were co-transfected with CoREST 3′ UTR–luc and pLKD.CMV.G&PR.U6-anti-miR-124 (anti-miR-124 group), pLKD.CMV.G&PR.U6 (Con-anti-miR-124 group), pLKD.CMV.GFP.U6 (Con-miR-124 group) or pLKD.CMV.GFP.U6-miR-124 (miR-124 group). After 48 h luciferase activity of each group was detected. *P<0.05 vs. Con-miR-124 group.

Fig. 4.

miR-124 targets CoREST 3′UTR to suppress its expression. Bioinformatics analysis of miR-124 binding site in 3′UTR of CoREST (A). Luciferase assays with the CoREST 3′ UTR luciferase reporter in RGC-5 cells (B). RGC-5 cells were co-transfected with CoREST 3′ UTR–luc and pLKD.CMV.G&PR.U6-anti-miR-124 (anti-miR-124 group), pLKD.CMV.G&PR.U6 (Con-anti-miR-124 group), pLKD.CMV.GFP.U6 (Con-miR-124 group) or pLKD.CMV.GFP.U6-miR-124 (miR-124 group). After 48 h luciferase activity of each group was detected. *P<0.05 vs. Con-miR-124 group.

Close modal

Establishment of rat chronic ocular hypertension glaucoma model

To determine whether axon of RGCs derived from Müller cells grew in vivo, we injected RGCs into rat chronic ocular hypertension glaucoma model. We found that the mean IOP of glaucomatous eyes significantly increased compared to that of controlateral eyes from day 3 to day 28 (Fig. 5A). IOP continued to increase and reached the maximum on 7-14 days, then decreased on day 28 and reached basic level 2 months later.

Fig. 5.

Establishment of rat chronic ocular hypertension glaucoma model. The mean IOP of glaucomatous eyes increased significantly compared with those of controlateral eyes from day 3 to day 28. *P<0.05 vs. corresponding control on the same day (A). TUNEL staining of apoptotic RGCs in retinal ganglion cell layer (B). IOP decreased to normal level, but the number of apoptotic cells still increased on day 60. *P<0.05 vs. day 7 (C).

Fig. 5.

Establishment of rat chronic ocular hypertension glaucoma model. The mean IOP of glaucomatous eyes increased significantly compared with those of controlateral eyes from day 3 to day 28. *P<0.05 vs. corresponding control on the same day (A). TUNEL staining of apoptotic RGCs in retinal ganglion cell layer (B). IOP decreased to normal level, but the number of apoptotic cells still increased on day 60. *P<0.05 vs. day 7 (C).

Close modal

By TUNEL assay we detected the apoptosis of RGCs in retinal ganglion cell layer (Fig. 5B). The proportion of TUNEL positive cells per section in different groups was 8.2±1.4%, 14.3±2.8%, and 19.2±2.8%, respectively. Moreover, the number of apoptotic cells gradually increased. Two months later, IOP decreased to basic level while the number of apoptotic cells continued to increase (Fig. 5C).

miR-124 promotes axon growth of RGCs differentiated from retinal stem cells in vivo

To determine whether miR-124 promotes axon growth of RGCs in vivo, differently treated RGCs were injected into rat chronic ocular hypertension glaucoma model. After 14 days, the eyeballs were extracted to detect the length and direction of ganglion cells axons. As shown in Fig. 6, Müller cells were stained as green by GS staining while RGCs were stained as red by Brn-3b staining. The differentiated RGCs migrated and proliferated between retinal layers in glaucoma rat models, and the axon budded from and extended around the cell body. The average axonal length was 501 µm in miR-124 group, markedly longer than control group and blank injection group (the mean axonal length was 220-230 µm). In contrast, the average axonal length was only 148 µm in anti-miR-124 group (Fig. 6A-E). Confocal scanning microscopy indicated that tracer CTb-594 was widely distributed in the large ganglion cell body and extended along the axon away from it (Fig. 6F).

Fig. 6.

miR-124 promotes axon growth of RGCs differentiated from retinal stem cells in vivo. Immunofluorescence staining and the average axonal length of the five groups: differentiated ganglion cells migrated and proliferated between retinal layers in glaucoma rat models, and the axon budded from and extended around the cell body. (A, B, C, D, E). Scale bar: 100 µm. Group A: cells infected by PGC-FU-Atoh7-GFP lentivirus only; Group B: cells with additional transfection with pLKD.CMV.GFP.U6; Group C: cells with additional transfection with pLKD.CMV.G&PR.U6; Group D: cells with additional transfection with pLKD. CMV.G&PR.U6-anti-miR-124; Group E: cells with additional transfection with pLKD.CMV.GFP.U6-miR-124. *P<0.05 vs. Group A. Confocal scanning microscopy showed that tracer CTb-594 was widely distributed in the large ganglion cell body and extended along the axon away from it (F).

Fig. 6.

miR-124 promotes axon growth of RGCs differentiated from retinal stem cells in vivo. Immunofluorescence staining and the average axonal length of the five groups: differentiated ganglion cells migrated and proliferated between retinal layers in glaucoma rat models, and the axon budded from and extended around the cell body. (A, B, C, D, E). Scale bar: 100 µm. Group A: cells infected by PGC-FU-Atoh7-GFP lentivirus only; Group B: cells with additional transfection with pLKD.CMV.GFP.U6; Group C: cells with additional transfection with pLKD.CMV.G&PR.U6; Group D: cells with additional transfection with pLKD. CMV.G&PR.U6-anti-miR-124; Group E: cells with additional transfection with pLKD.CMV.GFP.U6-miR-124. *P<0.05 vs. Group A. Confocal scanning microscopy showed that tracer CTb-594 was widely distributed in the large ganglion cell body and extended along the axon away from it (F).

Close modal

The axon of RGCs plays an important role in the establishment of synaptic connection to allow vision [17, 18]. Visual pathway between the eye and brain is constituted by axons from RGCs which can accurately make synaptic contacts with the target cells [19, 20]. Axonal projection capacity is mediated by growth cone at axon terminal, which is a highly initiative sensory structure with both outreach and retraction movement [21].

When axonal guidance molecules recognize and combine with growth cones receptors, downstream signaling pathways are activated, resulting in altered axonal growth direction. Recently, several ligand-receptor signal systems have been identified including Ephrins/Ephs, netrin/Dcc/UN-C5, Slits/Robos and Semaphorins/Neuropilins/ Plexins, which regulate the formation of axonal projection [22, 23]. Semaphorins are a group of secretory membrane-associated glycoproteins and Sema3A is the most important neural axonal guidance molecule [9, 15]. Sema3A plays an important role in axonal guidance, nerve fiber bundling, cell migration and adult neuronal damage repair. In the optic vesicle development, Sema3A is the most highly expressed protein. Sema3A in the retina induces axonal development and guidance by regulating neurite outgrowth and elongation as well as neurite outreach and retraction movement. NRP-1 has a high affinity for Sema3A and forms NRP-1/PlexinA receptor complex to regulate axonal growth and growth cone diversion.

CoREST is a cofactor of repressor element 1 silencing transcription factor (REST) and can repress NRP-1 transcription directly [15]. In this study, we found that miR-124 could upregulate NRP-1 expression in RGCs growth cones. In contrast, the depletion of miR-124 could downregulate NRP-1 expression in RGCs growth cone. By luciferase assay, we further confirmed that miR-124 directly targeted CoREST 3′UTR to suppress its expression, which could contribute to increased expression of NRP-1 and enhanced neurite growth and guidance.

Interestingly, as retinal Müller cell-derived stem cells differentiated, Sema3A and NRP-1 mRNA and protein expression decreased while CoREST and REST expression increased, leading to halted RGCs axon growth. Once exogenous miR-124 was introduced, Sema3A and NRP-1 expression increased while CoREST and REST expression decreased, and RGCs axons continued growth and the length reached almost 2, 000 µm. Furthermore, in rat chronic ocular hypertension glaucoma model, axon growth was the longest in miR-124 group and was the shortest in anti-miR-124 group.

Our in vitro and in vivo data provide the first evidence that miR-124 regulates axon growth of RGCs derived from Müller cells, and miR-124 has translational potential for gene therapy of glaucoma.

This study was supported by grant from National Nature Science Fund of China (No. 81400400 and 81770927).

All authors declare that they have no competing interests.

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