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Introduction

Retinal neovascularization (RNV) is the growth of new capillaries sprouting from the retinal veins and extending along the vitreous surface of the retina, which leads to vitreous hemorrhage, retinal detachment and even blindness (1–4). RNV plays an important role in many ocular diseases, such as proliferative diabetic retinopathy (PDR), retinal vein occlusion and retinopathy of prematurity (ROP). However, the pathological mechanisms involved in the formation and development of RNV remain unknown and still need to be further studied.

Slit-Robo signaling consists of three Slit (Slit1-3) secreted proteins and their four corresponding receptors [roundabout, axon guidance receptor, homologs 1–4 (Robo1-4)]. Slit-Robo signaling was first noted as acting as a repulsive cue in axonal guidance (5,6). Subsequently, the roles of Slit-Robo in organogenesis (7), neuronal migration (8) and tumor development (9–12) have been explored. Great progress has been made in studying the role of Robo1 signaling in angiogenesis, but the role of Robo1 in angiogenesis is still not fully understood. Han and Zhang demonstrated that Robo1 is involved in inhibiting corneal neovascularization (13). However, there is also evidence that Robo1 is pro-angiogenic: Wang et al (14) have reported that Robo1 mediates human umbilical vein endothelial cell (HUVEC) migration upon Slit2 stimulation, indicating that Slit2-Robo1 signaling promotes cancer angiogenesis. A previous study by Zhou et al (15) found that Robo1 was expressed in fibrovascular membranes (FVMs), demonstrating that Robo1 contributes to the development of diabetic retinopathy. The role of Slit-Robo signaling in RNV of oxygen-induced retinopathy (OIR) is researched in the present study.

MicroRNAs (miRNAs or miRs), small non-coding RNAs which are 21–24 nucleotides in length, act as important regulators of gene expression in mammals; several mammalian miRNAs have been identified as being located in the intronic regions of protein-encoding genes (host genes), termed intronic miRNAs (16). Intronic miRNAs can modulate the function of their host gene as the majority of them are coordinately expressed with the host genes (17,18). One example of endogenous RNA interference is when miRNAs negatively regulate gene translation by pairing with the 3′-UTR of a specific mRNA (19), and are involved in many functional regulatory processes (20–22). Many miRNAs have been found to play key roles in ocular neovascularization. Intraocular injection of certain miRNAs, such as miR-31 (21), miR-150 (23), miR-184 (23), miR-126 (24) and miR-410, has been shown to significantly inhibit retinal and choroidal neovascularization (25). miRNA-218 is an important intronic miRNA encoded by the Slit2 and Slit3 genes, which directly represses the expression of Robo1, Robo2 and contributes to retinal angiogenesis in mouse embryos (26). In order to investigate the effects of miR-218 on oxygen-induced RNV, a mouse model of OIR was established in the present study.

Materials and methods

Cell culture and transfection

Mouse retinal vascular endothelial cells were purchased from PriCells Biomedical Technology Co., Ltd. (Wuhan, China), and were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/l streptomycin (all from Gibco Life Technologies, Carlsbad, CA, USA) under conditions with 5% CO2, at 37°C, in a humidified incubator.

Cells in the exponential growth phase were seeded in 6-well plates (Corning Incorporated, Corning, NY, USA) at a density of 5×104 cells/ml. The cells were transfected with miR-218 mimic, miR-218 inhibitor, and the negative control (all from GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a 5:1 volume/mass ratio of reagent to oligodeoxynucleotide in serum-free M199 for 6 h. After transfection, the cells were incubated in complete DMEM. Transfection of the miR-218 mimics, miR-218 inhibitor and the negative control was performed using 1.0 μg of DNA per transfection. All transfections were performed according to the manufacturer's instructions.

siRNA design

Robo1 siRNA (sense, 5′-CGGGAAAGGCCG AGGAACAAAGGCAGC-3′ and antisense, 5′-GCUGCCUUU GUUCCUCGGCCUUUCCCG-3′) was synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

Plasmid construction

miR-218 was amplified using the following primers: miR-218 forward, 5′-TTCTGAGGATCCGTGGAGGCACCTTTTCCATA-3′ and reverse, 5′-ATTCTAAGATCTTTCACAGCTAGTCACACAATGG-3′. Plasmid vector pCDH-CMV-miR-218 was constructed by inserting a pri-miR-218 PCR fragment into the plasmid vector pCDH-CMV-MCS-EF1-Puro (Spiral Biotech, Inc., Norwood, MA, USA) through EcoRI and NotI digestion. The pCDH-CMV-mock vector was used as the negative control.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cells and retinal tissues using TRIzol reagent (Invitrogen). cDNA was synthesized, according to the manufacturer's instructions, with a cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan). qPCR was undertaken using SYBR-Green.

The primer sequences were as follows: miR-218, 5′-TTG TGCTTGATCTAACCATGT-3′; miR-218-1, 5′-CCATGGAA CGTCACGCAGC-3′; miR-218-2, 5′-GCGGAAAGCACCGTGCTC-3′; Slit2 forward, 5′-GCGCGTCTGGTGTGAAT GAA-3′ and reverse, 5′-CACAGTGGCACCAGGAGCAT-3′; Slit3 forward, 5′-TGGAAATACGCCTAGAACAG-3′ and reverse, 5′-ACCAGCGACGTGAGTGAT-3′; Robo1 forward, 5′-GAGGTAGCTATACTACGGGATGAC-3′ and reverse, 5′-CAGATGTAGTAGCCGACATCAGAC-3′; U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-AAAATATGGAACGCTTCACGA-3′.

Scratch wound assay

Cell migration ability was assessed using a scratch wound assay. Transfected cells were cultured in 6-well plates. When cells reached 90% confluence, a scratch wound was created using a pipette tip. Wound edges were photographed with a Nikon Eclipse TE20000-U (Nikon, Tokyo, Japan) and scratch widths were analyzed using ImageJ software (NIH). Each assay was completed in triplicate.

Mouse model of OIR

Neonatal CB57BL/6J mice were obtained from the Animal Institute of Chinese Academy of Medical Sciences. Neovascularization was induced as described by Kong et al (27). Briefly, at postnatal day 7 (P7), CB57BL/6J mice were exposed to hypoxic conditions (75% O2) for 5 days (P12) and then returned to room air in order to induce RNV.

The present study was approved by the Ethics Committee of Tianjin Eye Hospital (Heping, China). All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guide for the Care and Use of Laboratory Animals.

Intravitreal injection

Plasmids were packed using Lipof ectamine 2000 (Invitrogen) in accordance with the manufacturer's instructions. Twelve-day-old mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (30 mg/kg). Each animal received intravitreous injections of 0.4 μl (2 μg) pCDH-CMV-218 in one eye and 0.4 μl (2 μg) control plasmid [plasmid vector pCDH-CMV-MCS-EF1-Puro (Spiral Biotech, Inc.) without an insertion of miR-218] in the contra-lateral eye as the negative control. Intravitreal injections were performed at 11:00 a.m., and were administered to 1-mm posterior to the limbus of the eye using a 10-μl Hamilton syringe fitted with a 32G needle.

Western blotting

Total proteins of cells and retinas were isolated and separated on 10% SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Western blotting was performed according to standard protocols. α-tubulin was used as a loading control. The antibodies anti-Robo1 (ab7279), anti-Slit2 (ab7665), anti-Slit3 (ab11018) were from Abcam, and anti-α-tubulin (T5168) was from Sigma-Aldrich (St. Louis, MO, USA). Bands were quantified using ImageJ software (NIH).

Fluorescein angiography

On P17, mice (n=6/group) were anesthetized and perfused with fluorescein via retro-orbital injection of 2.5 mg/50 μl of FITC-dextran (Sigma-Aldrich), as previously described (28). Eyes were enucleated and fixed with 4% paraformaldehyde in PBS for 1 h. Retinas were then separated from the eyecup. Four incisions were made, and the retina was flat-mounted on a gelatin-coated slide. The vasculature was then examined under a fluorescence microscope (Nikon Eclipse TE20000-U). Images were analyzed using Photoshop 8.0 software (Adobe Systems, San Jose, CA, USA). Neovascularization was calculated as a ratio: the number of pixels in neovascular area to the total number of pixels in the retina.

Quantification of RNV

At p17, the eyes of the mice (n=6/group) were enucleated and fixed with 10% formaldehyde and embedded in paraffin. Sagittal sections (6-μm-thick) were made through the cornea parallel to the optic nerve, and then they were stained with hematoxylin and eosin (H&E). The nuclei of vascular cells on the vitreal side of the retina were counted under a light microscope. Ten non-continuous sections from each eye were examined, and numbers of cells were averaged in each group. The average number of pre-retinal vascular nuclei was compared.

Statistical analysis

Data are expressed as the means ± standard deviation (SD) and were analyzed using SPSS 11.5 (SPSS, Inc., Chicago, IL, USA). To compare multiple sets of data, one-way analysis of variance (ANOVA) was used. For paired data sets, the LSD t-test was used. Bivariate correlations were calculated by Spearman's rank correlation coefficients. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

Inhibition of cell migration by miR-218 is mediated by Robo1

The cell migration ability of each group is shown in Fig. 1: overexpression of miR-218 caused by miR-218 mimic significantly reduced endothelial cell (EC) migration, whereas inhibition of miR-218 expression using the miR-218 inhibitor markedly promoted EC migration (Fig. 1A and B).

Figure 1 miR-218 inhibits endothelial cell (EC) migration by suppressing roundabout, axon guidance receptor, homolog 1 (Robo1) expression. (A) Quantification of cell migration. (B) Representative images of cell migration in the wounded area. (C) Robo1 expression was determined by western blotting and normalized to the internal control α-tubulin. *P<0.05.

Robo1 expression of each group is shown in Fig. 1C. The results indicate that upregulation of miR-218 expression not only reduced EC migration but also decreased Robo1 expression. Conversely, downregulation of miR-218 expression using the miR-218 inhibitor increased Robo1 expression and, as was noted previously, promoted EC mobility. However, as shown in Fig. 1A, miR-218 inhibitor did not promote the migratory ability of ECs after Robo1 knockdown by siRobo1. These observations suggest that miR-218 suppresses EC migration by inhibiting Robo1 expression.

miR-218 and Robo1 expression in RNV of mice with OIR

The expression level of miR-218 in the retinas of OIR mice was detected. As shown in Fig. 2A, RT-qPCR results demonstrated that the expression level of miR-218 was significantly decreased (P<0.05) in retinas of mice with OIR at P17. We then compared mRNA and protein expression levels of Robo1 in retinas of mice with OIR with those of the control mice. The mRNA and protein levels of Robo1 were markedly upregulated at P17 in mice with OIR (Fig. 2).

Figure 2 Identification of decreased miR-218 and increased roundabout, axon guidance receptor, homolog 1 (Robo1) in retinas from the rat model of oxygen- induced retinopathy (OIR). (A) RT-qPCR showed that miR-218 was significantly decreased in the model of OIR compared to normal (control) mice (n=5). However, the expression of Robo1 mRNA was significantly increased (n=6). (B) Western blot analysis demonstrated that Robo1 protein was highly expressed in retinas of mice with OIR compared to normal (control) mice. *P<0.05.

Role of miR-218 coding genes (miR-218-1 and miR-218-2) and corresponding host genes (Slit2 and Slit3)

To determine which miR-218 coding gene is involved in downregulation of miR-218 in mice with OIR, we evaluated miR-218-1, miR-218-2, miR-218, Slit2 mRNA and Slit3 mRNA expression in retinas of mice with OIR by RT-qPCR. As shown in Fig. 3B–E, statistical analysis revealed that a positive correlation existed between miR-218-1 and Slit2, miR-218-2 and Slit3, as well as miR-218-1 and miR-218. There was no correlation, however, between miR-218-2 and miR-218. This meant that in the retinas of the mice with OIR, miR-218-1 and miR-218-2 were co-transcribed with their host gene respectively and that downregulation of miR-218 was caused by decreased expression of miR-218-1, not miR-218-2. When mRNA levels of Slit2 and Slit3 in retinas from control mice and mice with OIR were compared, mRNA expression of Slit2 in retinas of mice with OIR was 0.48-fold less (P<0.05) than in the retinas of the normal control mice; mRNA expression of Slit3 in retinas from mice with OIR was 1.12-fold greater than in the control mice although this was not statistically significant (P>0.05) (Fig. 3A). Similar results were also observed in protein expression levels of Slit2 and Slit3 (Fig. 3F and G). The results suggest that decreased expression of miR-218-1 and its host gene Slit2 is involved in RNV in OIR.

Figure 3 Expression of miR-218, miR-218-1, miR-218-2, Slit2 and Slit3 in retinas in mice with oxygen-induced retinopathy (OIR) and control mice, as assessed by RT-qPCR. (A) Expression levels of miR-218, miR-218-1 and Slit2 were significantly decreased in OIR mice compared to normal mice. However, miR-218-2 and Slit3 were slightly elevated in OIR mice. (B and C) Positive correlations were observed between miR-218-1 and Slit2 as well as between miR-218-2 and Slit3. (D and E) Positive correlations were noted between miR-218-1 and miR-218. However, no significant positive correlation was noted between miR-218-2 and miR-218. (F) Representative images of western blotting of Slit2 and Slit3 in mice with OIR and control mice. (G) Western blotting indicated that Slit2 protein expression significantly decreased (P<0.05). However, Slit3 protein expression was slightly elevated (P>0.05) in mice with OIR. *P<0.05, **P<0.01.

miR-218 inhibits RNV through suppressing Robo1 expression

A plasmid pCDH-CMV-miR-218 expressing miR-218 and a negative control plasmid pCDH-CMV-mock expressing mock sequence were constructed. Intravitreal injection of plasmids was performed at P12.

At P17, we analyzed mRNA levels of miR-218 and Robo1 after intravitreal injection to verify whether plasmids reached the retina. RT-qPCR results showed that pCDH-CMV-miR-218 significantly increased the expression of miR-218 and reduced that of Robo1 in retinas (Fig. 4A). Western blotting indicated that intravitreal injection of pCDH-CMV-miR-218 significantly decreased the protein level of Robo1 in the retinas of mice with OIR (Fig. 4B). However, pCDH-CMV-mock had almost no effect on the expression of miR-218 and Robo1.

Figure 4 Downregulation of roundabout, axon guidance receptor, homolog 1 (Robo1) expression by intravitreal injection of miR-218 plasmid in mice with oxygen-induced retinopathy (OIR). (A) RT-qPCR results showed that relative Robo1 expression in the miR-218-transfected group was significantly decreased compared to the negative control group and OIR group. (B) Western blotting for Robo1 expression before and after miR-218 intravitreal injection. Statistical analysis of western blotting indicated that intravitreal injection of miR-218 effectively inhibited Robo1 protein expression in retinas in mice with OIR. n=6 mice/group. *P<0.05.

To examine the effects of miR-218 on RNV, we evaluated the retinal vasculature at P17 by fluorescein angiography in the flat-mounted retinas. In retinas of normal mice we noted a mature capillary network that extended from the optic to the periphery (Fig. 5A1). The retinas of mice in the OIR and pCDH-CMV-mock groups displayed more neovascular tufts (Fig. 5A2 and A3). By contrast, the retinas of the pCDH-CMV-miR-218 group exhibited lesser neovascularization (Fig. A4). The RNV was quantified by measuring areas of new blood tufts in the whole mounted retina. The results showed that a large number of neovascular tufts appeared in the OIR group [neovascularization (NV), 38.9% of whole retina] and pCDH-CMV-mock group (NV, 36.8% of whole retina). However, compared to the OIR group, the neovascularized area was significantly decreased in the pCDH-CMV-miR-218 group (NV, 18.6% of whole retina) (Fig. 5B). No neovascularization was observed in the normal group. These results revealed that miR-218 exerts an anti-neovascularizing effect on RNV in mice with OIR.

Figure 5 Representative retinal angiographs show that miR-218 effectively inhibits retinal neovascularization (RNV) in mice with oxygen-induced retinopathy (OIR) (magnification, x40). (A1) Retinal vessels in the normal mice. (A2 and A3) In the OIR group and pCDH-CMV-mock (negative control) group, tortuous and dilated retinal vessels were observed, and neovascular tufts were present at the junction between perfused and non-perfused regions (red arrows). (A4) After miR-218 injection, neovascular tuffs and non-perfused area were reduced. (B) Quantification of RNV from the group with OIR, pCDH-CMV-mock (negative control) group and miR-218-injected group (pCDH-CMV-miR-218). RNV was assessed using the ratio of the neovascular tuft area to total retinal area, and measured by image-analysis software. n=6 mice/group. *P<0.05.

To further study the inhibitory effect of miR-218 on angiogenesis, histological analysis was performed. We counted the vascular cell nuclei which broke through the inner limiting membrane (ILM), a marked feature of OIR (29). There were no neovascular nuclei in the normal group (Fig. 6A1), and the average number of vascular cell nuclei was significantly increased in the OIR group and pCDH-CMV-mock group, 61.48±6.92 and 58.98±6.48, respectively (Fig. 6A2 and A3). However, in the pCDH-CMV-miR-218 group we noted less neovascularization (Fig. 6A4) (12.64±1.42 pre-retinal cells/section), indicating that restoration of miR-218 played an inhibitory role in RNV in OIR (Fig. 6B).

Figure 6 Hematoxylin and eosin (H&E) staining of neovascularization in mouse retinas (magnification, x40). At P17, the retinas were fixed and stained. (A1) Normal group; (A2) OIR group; (A3) pCDH-CMV-mock group (negative control); (A4) miR-218 group. Arrows indicate pre-retinal vascular cells; (B) Vascular cell nuclei broke through the inner limiting membrane (ILM) (as indicated by arrows) and were counted in ten sagittal sections and averaged. The miR-218 group exhibited less neovascularization. n=6 mice/group. *P<0.05.NS, not significant.

Taken together, these results indicate that miR-218 inhibited retinal angiogenesis in OIR by suppressing Robo1 expression.

Discussion

Robo1, a member of the Robo receptor family, is involved in signaling in the nervous system (30). However, Robo1 plays a positive role in the migration of endothelial cells. Previous research using monkey choroidal retinal endothelial cells (31) and HUVECs (14) has demonstrated that decreased Robo1 markedly inhibited EC migration, and this conclusion was further confirmed using mouse retinal endothelial cell in this study.

In the present study, we noted that in vitro cell migration was markedly suppressed when the miR-218 mimic was transfected into ECs. Moreover, the expression of Robo1 decreased in cells. Additionally, a negative expression pattern was noted when miR-218-inhibitor was transfected into ECs: Robo1 expression increased with the decrease of miR-218 expression, and EC migration increased significantly. However, miR-218 inhibitor and siRobo1 had almost no effect on EC migration. Our results indicated that the inhibitory effect of miR-218 on EC migration was mediated by Robo1.

The data presented in the present study indicate that mRNA and protein expression levels of Robo1 were significantly elevated in retinas of mice with OIR. This result was consistent with that of a previous study (31) and substantiated the potential role of Robo1 in RNV. Retinal vascular formation was also observed, suggesting that there was a significant positive correlation between Robo1 overexpression and RNV. Investigation of tumors indicated that Robo1 play a part in angiogenesis (14). Previous studies have suggested that Robo1 is involved in ocular neovascularization (31,32). Genetic evidence provided by Rama et al supported a negative role for Robo1 in ocular pathological neovascularization (33). Our results also verified this point. In the present study, we found that retinal neovascularization and endothelial cells breaking into the inner nuclear layer in the retinas were both significantly reduced following the administration of pCDH-CMV-miR-218. Therefore, we suggest that downregulation of Robo1 significantly suppresses RNV.

In our study, a positive correlation between pre-miR-218 and their host gene was observed, demonstrating that miR-218-1 and miR-218-2 co-express with their host genes during RNV in OIR. Furtherore, a significant correlation between the expression of miR-218 and miR-218-1 was observed, indicating that the downregulation of miR-218-1 leads to the downregulation of miR-218. In addition, the expression of miR-218-1 and its host gene, Slit2 (not miR-218-2 and Slit3), were concomitantly downregulated during RNV in mice with OIR, indicating that miR-218-1 and Slit2 are involved in RNV by negatively regulating retinal angiogenesis.

As an intronic miRNA, miR-218 is involved in ligand/receptor signaling. In brief, miR-218 is located in and is co-expressed with its host gene, Slit, whose receptor gene, Robo1, is one of the targets of miR-218. Thus, Slit-miR-218-Robo signaling is established. Ligand/receptor signaling transduction mediated by intronic miRNAs has also demonstrated in other studies. miR-338, also an intronic miRNA, was shown to silence the target gene, which was antagonistic to its host gene, AATK (37). Tie et al (34) pointed out that miR-218 mediated Slit/Robo signaling, forming a negative feedback circuit, inhibiting gastric cancer metastasis. The Slit-miR-218-Robo regulatory network (26) was also found to be essential for the normal vascularization of the retina. In our study, RNV in mice with OIR was shown to be regulated by the Slit-miR-218-Robo axis. We thus speculated that the regulation of Slit-miR-218-Robo signaling may provide a means of suppressing RNV by preventing the overactivation of ligand/receptor signaling.

In the mice with OIR, we noted that the expression of miR-218 significantly decreased, and the expression of Robo1 increased. The restoration of miR-218 inhibited retinal angiogenesis, suggesting that miR-218 is an important regulator of RNV. Our results also suggest that miR-218 inhibits RNV through mediating the downregulation of Robo1 expression.

In conclusion, the Slit-miR-218-Robo axis plays a role in retinal angiogenesis. Slit2 interacts with Robo1 to inhibit RNV mediated by miR-218. miR-218 may thus be a therapeutic candidate for the targeted treatment of RNV

Acknowledgments

This study was supported by the Natural Science Foundation of Tianjin City (no. 13JCYBJC22900).

References