Down-Regulation of MicroRNA-184 Is Associated With Corneal Neovascularization

Abstract

Purpose: Although microRNA-184 (miR-184) is abundantly expressed in the corneas, the role of miR-184 in corneal neovascularization remains unknown. Here we investigated the association between miR-184 expression and corneal neovascularization.

Methods: Quantitative real-time PCR assay was conducted to detect the expression of miR-184 and its potential target genes in the corneal epithelium of rats with corneal suture-induced neovascularization. MicroRNA-184 was also applied topically to the suture rats. Mimic and inhibitor of miR-184 were transfected into the cultured human umbilical vein endothelial cells (HUVECs), human corneal epithelial (HCE) cells, and simian choroidal endothelial cells (RF/6A). The following experiments were performed to evaluate the effects of miR-184 in these transfected cells: cell proliferation by cell viability assay, cell migration by a scratch wound test, VEGF-induced tube formation, and VEGF and β-catenin levels by Western blot analysis.

Results: The expression of miR-184 was significantly reduced, whereas the gene expression of frizzled-4, a receptor of the Wnt pathway, was up-regulated in the corneal epithelium of corneal suture rats. The corneal neovascularization induced by suture was ameliorated by topical administration of miR-184. In the cells transfected with mimic and inhibitor of miR-184, miR-184 significantly suppressed the cell proliferation and cell migration of HUVECs, miR-184 down-regulated VEGF, and β-catenin expression in HUVECs and HCE cells. Furthermore, miR-184 inhibited the tube formation of RF/6A cells.

Conclusions: Down-regulation of miR-184 is associated with up-regulation of VEGF and Wnt/β-catenin expression as well as corneal neovascularization, indicating that miR-184 negatively regulates corneal neovascularization.

MicroRNAs are a group of endogenous, highly conserved, and short (18–25 nucleotides long) noncoding RNAs that regulate the gene expression at the posttranscriptional level and consequently confer biological functions. A number of studies have demonstrated that microRNAs are associated with various physiological and pathologic functions.^1,2 Multiple target genes regulated by microRNAs have been identified.^3,4 

Under normal conditions, the cornea is transparent and avascular. On the other hand, corneal angiogenesis or neovascularization occurs during corneal or ocular surface diseases, such as infections, pterygium, and chemical injury. However, the mechanisms responsible for the avascularity in normal cornea and transitions to corneal neovascularization in eye diseases have not been fully understood. It has been reported that there are several important factors regulating corneal angiogenesis or neovascularization, including proangiogenic factors such as VEGF and antiangiogenic factors such as pigment epithelium–derived factor (PEDF). Recent results suggest that microRNAs also might play vital roles in modulating ocular angiogenesis or neovascularization.^5–8 

MicroRNA-184 (miR-184) serves as a mediator in apoptosis and neural development.⁹ It has been reported that miR-184 is abundantly expressed in the cornea¹⁰ and that there is an inverse relationship between miR-184 levels and corneal disease progression. This association occurs in keratoconus and some other eye diseases.^11–13 A recent report shows that miR-184 targets or regulates the Wnt signaling pathway in the retina.¹⁴ However, the role of miR-184 in the corneal avascularity or corneal neovascularization remains unknown. 

We recently applied a corneal suture technique in rats and examined the effects of a new treatment on corneal neovascularization. We demonstrated that corneal suture can induce corneal neovascularization and inflammation and proved that the corneal suture can be used as a good and specific model for corneal neovascularization.¹⁵ In addition, we also showed that corneal suture increased levels of VEGF and key factors of the Wnt pathway, such as β-catenin.¹⁵ In this present study, we expanded on the preceding by investigating the role of miR-184 in corneal neovascularization using the same corneal suture model, and we also performed transfection of mimics and inhibitor of miR-184 in cultured cells to further illustrate the underlying mechanisms. 

Materials and Methods

Materials

The miRNA mimics or inhibitors were purchased from Life Technologies Corp. (mirVana; Carlsbad, CA, USA). The transfection reagent was purchased from Qiagen (HiPeFect; Hilden, Germany). The cell viability assay kits were purchased from Dojindo Co. (CCK-8; Tokyo, Japan). The basement membrane matrix was purchased from Becton, Dickinson and Company (Matrigel; Franklin Lakes, NJ, USA). The antibody of anti–β-catenin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The antibody of anti-VEGF was purchased from Abcam (Cambridge, MA, USA) and the antibody of anti–β-actin from Sigma-Aldrich Corp. (St. Louis, MO, USA). Recombinant human VEGF165 protein was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). 

Corneal Suture Model

Wistar rats (male, weighing from 180 to 220 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and were kept in an air-conditioned standard facility. Food and water were available ad libitum. All of the experiments with rats were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the experimental protocol was approved by the experimental animal ethics committee of Xiamen University (Xiamen, Fujian, China). The rat corneal suture procedure was described previously.¹⁵ Briefly, after induction of general anesthesia by intraperitoneal injection with pentobarbital (40–50 mg/kg), topical application of 0.5% proparacaine ophthalmic solution was added. Three corneal sutures (10-0 nylon) were conducted in the cornea between corneal center and the limbus at the 12, 4, and 8 o'clock positions. The rats were carefully maintained, and the eyes were observed daily after surgery. After 7 days, all rats were euthanized, and the whole corneas were dissected for quantitative real-time PCR assay following the method described below, in addition to other experiments and measurements reported previously.¹⁵ Five rat corneal samples were used for each control and suture group in this present study. 

In Vivo Experimental Procedure

The topical administration of miR-184 was applied in the corneal suture rats. Twenty rats were randomly divided into four groups (n = 5 in each group): (1) normal or control group without suture and any treatment; (2) suture plus saline group; (3) suture plus miRNA mimic negative control (mirVana; Ambion, Austin, TX, USA) (0.5 mM) group (or scramble RNA group); and (4) suture plus miR-184 mimic (Ambion) (0.5 mM) group. The miRNA was dispersed in transfection regent (Lipofectamine RNAiMAX; Invitrogen, Carlsbad, CA, USA) following manufacturer's instruction. All topical administration was at 10 μL per eye each time and given once 30 minutes after suture and thereafter twice a day (9 AM and 5 PM) on days 1, 3, and 5 after suture. Approximately 50 pmole miRNA was administered each time. The rats were carefully maintained, and the eyes were observed daily after surgery. During the experiment, the clinical evaluation of corneal neovascularization was performed by a single masked, experienced ophthalmologist using slit-lamp microscopy on days 4 and 7. Images of corneal neovascularization were captured using a slit-lamp microscope, and the method of evaluation of corneal neovascularization was reported previously.¹⁵ 

Quantitative Real-Time RT-PCR for MicroRNA and mRNA Expression Analysis

Total RNA was extracted from the corneas with reagent (TRIzol; Invitrogen). Complementary DNA (cDNA) was prepared using TaqMan MicroRNA Assay Kits (Applied Biosystems, Carlsbad, CA, USA) from total RNA (100 ng), including microRNAs and specific stem-loop primers for miR-184 or small nuclear RNA U6 (an endogenous small nuclear RNA [snRNA]) and a cocktail containing reverse transcription buffer, dNTP mix, recombinant RNase inhibitor, and reverse transcriptase (Multiscribe; Applied Biosystems). Real-time PCR was performed on an Applied Biosystems StepOne Real-Time PCR System. Amplification of a single fragment was confirmed by a dissociation curve with good correlation with standards and threshold-cycle values. Gene expression was evaluated by an assay (SYBR Green; TaKaRa, Otsu, Japan) or semiquantitative RT-PCR with Friz4 mRNA special primers: 5′-CTTCCCACTGTGTGGACCTT-3′ (forward), 5-CTATCCCTCGAACGCAAGTC-3′ (reverse). The PCR reactions were performed on a real-time system (BIO-RAD CFX-96 system; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds, followed by melting curve analysis. 

Cell Culture Procedures

For primary HUVEC isolation, fresh human umbilical cord veins were digested with 0.25% EDTA trypsin (Invitrogen) as described previously with modifications.¹⁶ This investigation was carried in accordance with the Declaration of Helsinki for medical research involving human subjects. All tissue donors signed a written informed consent, and the Institutional Medical Ethics Committee of Xiamen University approved the study protocol. The endothelial cells were dispersed to single cells and cultured in M200 medium supplemented with low serum-growth supplement and 1% penicillin. The cultured HUVECs obtained from passages 3 and 6 were used in all the experiments. 

A cell line derived from HCE cells, simian virus-40 transformed, was obtained from RIKEN Biosource Center (Tokyo, Japan). The HCE cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (F-12; Invitrogen) containing 6% fetal bovine serum, 7.5 mg/mL insulin, and 10 ng/mL epidermal growth factors. 

Simian choroidal endothelial cells (RF/6A) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The RF/6A cells were cultured in DMEM with 10% fetal bovine serum (Gibco; Invitrogen, Grand Island, NY, USA), 100 units/mL penicillin at 37°C under 5% CO₂, and 95% humidified air. 

Cell Transfection Protocol

To analyze the impacts of miRNA in the cultured cells, the mirVana miRNA mimic negative control (25 nM), miR-184 mimic (25 nM), mirVana miRNA inhibitor negative control (50 nM), or miR-184 inhibitor (50 nM; Ambion) were transfected separately into HUVECs or HCE cells using transfection reagent (Hiperfect; Qiagen) following the manufacturer's instruction. At 24 to 48 hours post transfection, the cells were harvested, and the protein and RNA levels of identified target genes were analyzed by Western blot analysis and quantitative real-time PCR assay, respectively. 

Cell Viability Assay

To determine transfected cell viability, the CCK8 cell viability assay was used as described in our previous study.¹⁵ Cells were seeded (1 × 10⁴/well) onto a 96-well plate and were allowed to attach overnight. After 24 hours, the cells were transfected by a miR-184 mimic or miR-184 inhibitor. At 48 hours post transfection, a cell viability test was performed, and culture media containing 10% CCK8 reagent was added to each well. The plate was incubated for 4 hours at 37°C to produce a color reaction. Absorbance was measured with a microplate reader (BioTek EXL800; BioTek Instrument, Inc., Highland Park, Winooski, VT, USA) at 570 nm. 

Cell Migration Assay

A scratch wound test was conducted to detect the cell migration of HUVECs. After the cells were transfected for 48 hours by a miR184 mimic or a miR184 inhibitor, a scratch was applied in the center of the culture dish. DAPI (4′,6-diamidino-2-phenylindole) was added and incubated for 3 minutes before the image was grabbed. The cell images were recorded at 0 and 48 hours. The migration area was measured and analyzed to represent the cell migration of HUVECs. 

Tube Formation Assay

To evaluate VEGF activity and angiogenesis, a tube formation assay was performed with transfected RF/6A cells. Briefly, the Matrigel Matrix (BD Biosciences, Bedford, MA, USA) was aliquoted (100 μL/well) into a 96-well plate and polymerized for 30 minutes at 37°C. Prior to seeding, the transfected cells were collected with trypsin digestion, and cell density was quantified using a hemocytometer. Cells were seeded at a density of 1.5 × 10⁴ cells per well in M200 containing 20 ng/mL VEGF₁₆₅. After 6 hours of incubation in a 5% carbon dioxide cell incubator, the cells were photographed using an inverted microscope (magnification ×40; Olympus, Tokyo, Japan). The perimeter of tube formation by RF/6A cells was measured and analyzed by ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated three times. 

Western Blot Analysis

Total proteins of the corneas or cells were extracted with cold radioimmunoprecipitation assay buffer. Equal amounts of proteins were subjected to electrophoresis on 8% SDS-PAGE and then electrophoretically transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 1% BSA for 1 hour, then subsequently incubated overnight at 4°C with primary antibodies of β-catenin (1:1000), VEGF (1:1000), and β-actin (1:10,000) as loading control. After three 10-minute washes with Tris-buffered saline with Tween, the membrane was incubated for 1.5 hours with a 1:5000 dilutions of an horseradish peroxidase–conjugated anti-rabbit IgG antibody (Bio-Rad Laboratories, Inc.) for 1 hour. The specific bands were detected by enhanced chemiluminescence reagents (Lulong, Inc., Xiamen, China) and recorded by transilluminator (ChemiDox XRS; Bio-Rad Laboratories, Inc.). 

Statistical Analysis

Data of quantitative real-time PCR assay between control group and suture group were analyzed by a Student's t-test. Data of in vivo corneal neovascularization evaluation were analyzed by 1-way ANOVA, followed by a post hoc analysis Tukey test to compare the differences between the groups. Data of CCK-8 cell viability assay, cell migration, tube formation, and Western blotting between the scramble RNA group and mimic/inhibitor of miR-184 groups were also analyzed by a Student's t-test. A value of P < 0.05 was considered statistically significant. 

Results

Corneal Suture Induces miR-184 Down-Regulation and Frizzled-4 Up-Regulation

The rat corneal suture model was used to further investigate the pathogenesis of corneal neovascularization by focusing on the roles miR-184, which is abundantly expressed in the cornea¹⁰ and the potential target genes of miR-184. We conducted quantitative real-time PCR assay to detect the expression of miR-184 in the corneal epithelium after corneal suture surgery. It was demonstrated that the expression levels of miR-184 were significantly reduced by approximately 75% in the corneal epithelium of suture rats compared with control ones (Fig. 1A). 

Figure 1

 

Corneal suture-induced down-regulation of miR-184 expression and up-regulation of Frz4 gene expression. Real-time PCR assay was conducted in the corneal epithelium of rats 7 days after corneal suture surgery. (A) Comparison of expression of miR-184 between the control group and suture group. (Data are presented as mean ± SEM, n = 5 in each group, ***P < 0.001.) (B) Comparison of gene expression of Frz4, a member of frizzled receptors of the Wnt pathway, between the control group and suture group. (Data are presented as mean ± SEM, n = 5 in each group, **P < 0.01.)

Several microRNAs contribute to angiogenesis regulation via VEGF modulation.^17–19 Here, we revealed by real-time PCR assay that the expression of frizzled-4 (Frz4), a member of frizzled receptor of the Wnt signaling pathway, was also up-regulated in the corneal epithelia of rats after corneal suture surgery, compared with that in control rats (Fig. 1B). 

Taken together, the preceding results suggest that down-regulation of miR-184 and up-regulation of its potential targets VEGF and Wnt/β-catenin signaling contribute to the corneal neovascularization in this corneal suture model. 

MicroRNA-184 Ameliorates Corneal Neovascularization in Sutured Rats

To verify the association of miR-184 and evaluate the potential efficacy of miR-184 on the corneal neovascularization, we applied topical administration of miR-184 to corneal sutured rats. It was demonstrated that miR-184 significantly ameliorated the corneal neovascularization of sutured rats on day 7 after suture, compared to the sutured group and the scramble treated group (Figs. 2A–E). 

Figure 2

 

MicroRNA-184 ameliorated corneal neovascularization in sutured rats. (A–D) Representative micrographs of corneal neovascularization on day 7 after corneal suture. (A) Group of normal or control rats without suturation or any treatment; (B) group of suturation plus saline; (C) group of suturation plus mirVana miRNA mimic negative control (or scrambled control group); (D) group of suturation plus miR-184. The new vessels were seen in corneas (B and C), and the new vessels were reduced in (D) (arrows). (E) The statistical comparison of the data of neovascularization among the above four groups on day 4 and day 7. (Data are presented as mean ± SEM, n = 6–9 in each group, **P < 0.01, ***P < 0.001.)

MicroRNA-184 Suppresses Cell Proliferation and Cell Migration of HUVECs

To broaden our understanding of the functional significance of miR-184 expression, we transfected miR-184 mimic and inhibitor into cultured HUVECs and evaluated their effects on cell proliferation and cell migration of HUVECs. 

We measured the expression levels of miR-184 of HUVECs by real-time PCR assay after transfection for 24 hours to verify if these miR-184 modifications were successfully transfected into the cultured HUVECs. It was shown that the expression of miR-184 mimic was significantly increased compared to the scramble. (Data not shown.) As shown in Figure 3A, the miR-184 mimic significantly decreased the cell viability of HUVECs after transfection for 48 hours compared with the scrambled RNA group. In contrast, the miR-184 inhibitor increased the cell viability of HUVECs compared to its scrambled counterpart (Fig. 3B). Furthermore, we demonstrated that the miR-184 mimic significantly inhibited the cell migration of HUVECs after transfection for 48 hours compared with the scrambled group (Figs. 3C, 3D); on the other hand, the miR-184 inhibitor increased the cell migration of HUVECs compared to its scrambled counterpart (Figs. 3E, 3F). 

Figure 3

 

MicroRNA-184 suppressed cell proliferation and cell migration of HUVECs. The CCK-8 assay was performed to measure the cell viability or proliferation, and a scratch wound test was conducted to detect the cell migration. (A) Statistical analysis of cell viability of the cultured HUVECs transfected with scramble or the mimic of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, ***P < 0.001.) (B) Statistical analysis of cell viability of the HUVECs transfected with scrambled or the inhibitor of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, ***P < 0.001.) (C) Representative images of the migration area of HUVECs transfected with scrambled or the mimic of miR-184 for 48 hours (blue, DAPI nuclear staining). Scale bar: 100 μm. (D) Statistical analysis of cell migration of the HUVECs transfected with scrambled or the mimic of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, ***P < 0.001.) (E) Representative images of the migration area of HUVECs transfected with scramble or the inhibitor of miR-184 for 48 hours (blue, DAPI nuclear staining). Scale bar: 100 μm. (F) Statistical analysis of cell migration of the cultured HUVECs transfected with scrambled or the inhibitor of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, ***P < 0.001.)

These results indicate that miR-184 expression modulation also affects control of HUVEC proliferation and migration as well as neovascularization. 

MicroRNA-184 Negatively Regulates VEGF and β-Catenin in HUVECs and HCE Cells

We next investigated the roles of miR-184 in mediating control of the expression of two of its potential gene targets: VEGF and β-catenin in the cultured HUVECs and HCE cells transfected with the miR-184 mimic and inhibitor. 

We first performed Western blot to detect the changes of VEGF and β-catenin of the transfected HUVECs, and we showed that the miR-184 mimic significantly down-regulated protein levels of VEGF and β-catenin of HUVECs after transfection for 48 hours, compared with the scrambled group (Figs. 4A, 4B, 4E, 4F). In contrast, the miR-184 inhibitor up-regulated the levels of VEGF and β-catenin of HUVECs compared to its scrambled counterpart (Figs. 4C, 4D, 4G, 4H). 

Figure 4

 

MicroRNA-184 down-regulated the levels of VEGF and β-catenin of HUVECs. Western blot analysis was performed to detect protein levels of VEGF and β-catenin in the transfected cultured HUVECs. (A) Representative images of Western blotting of VEGF in the HUVECs transfected with scramble or the mimic of miR-184 for 48 hours. (B) Statistical analysis of Western blotting data of VEGF of the cultured HUVECs transfected with scrambled or the mimic of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 4 in each group, **P < 0.01.) (C) Representative images of Western blotting of VEGF in the HUVECs transfected with scramble or the inhibitor of miR-184 for 48 hours. (D) Statistical analysis of Western blotting data of the HUVECs transfected with scramble or the inhibitor of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 4 in each group, **P < 0.01.) (E) Representative images of Western blotting of β-catenin in the HUVECs transfected with scramble or the mimic of miR-184 for 48 hours. (F) Statistical analysis of Western blotting data of β-catenin in the HUVECs transfected with scramble or the mimic of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 4 in each group, ***P < 0.001.) (G) Representative images of Western blotting of β-catenin in the HUVECs transfected with scramble or the inhibitor of miR-184 for 48 hours. (H) Statistical analysis of Western blotting data of β-catenin the HUVECs transfected with scramble or inhibitor of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 4 in each group, **P < 0.01.)

We also measured the miR-184 expression levels of HCE cells by real-time PCR assay after transfection for 24 hours to verify that miR-184 was successfully transfected in the cultured cells. We showed that the expression level of miR-184 mimic was dramatically increased compared to the scrambled group. (Data not shown.) We next examined the alterations of VEGF and β-catenin by Western blot analysis in the transfected HCE cells, and we revealed that the miR-184 mimic significantly down-regulated protein levels of VEGF and β-catenin of HCE cells after transfection for 24 hours compared with the scrambled group (Figs. 5A, 5B, 5E, 5F). On the other hand, the miR-184 inhibitor up-regulated the levels of VEGF and β-catenin of HCE cells, compared with those in its scrambled counterpart (Figs. 5C, 5D, 5G, 5H). 

Figure 5

 

MicroRNA-184 down-regulated the levels of VEGF and β-catenin of HCE cells. Western blotting was performed to detect protein levels of VEGF and β-catenin in the transfected HCE cells. (A) Representative images of Western blotting of VEGF in the HCE cells transfected with scramble or the mimic of miR-184 for 48 hours. (B) Statistical analysis of Western blotting data of VEGF of the HCE cells transfected with scramble or the mimic of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, **P < 0.01.) (C) Representative images of Western blotting of VEGF in the HCE cells transfected with scramble or the inhibitor of miR-184 for 48 hours. (D) Statistical analysis of Western blotting data of VEGF of the HCE cells transfected with scramble or the inhibitor of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, ***P < 0.001.) (E) Representative images of Western blotting of β-catenin in the HCE cells transfected with scramble or the mimic of miR-184 for 48 hours. (F) Statistical analysis of Western blotting data of β-catenin of the HCE cells transfected with scramble or the mimic of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 5 in each group, **P < 0.01.) (G) Representative images of Western blotting of β-catenin in the HCE cells transfected with scramble or the inhibitor of miR-184 for 48 hours. (H) Statistical analysis of Western blotting data of β-catenin of the HCE cells transfected with scramble or the inhibitor of miR-184 for 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, *P < 0.05.)

These results suggest that miR-184 negatively regulates both VEGF and β-catenin, the key effector in the Wnt signaling pathway. 

MicroRNA-184 Inhibits Endothelial Cell Tube Formation

Finally, we performed a VEGF-induced tube formation model in vitro to identify the effects of miR-184 on tube formation in the cultured simian choroidal endothelial cells (RF/6A) transfected with the miR-184 mimic and inhibitor. After 48 hours of transfection, it was demonstrated that the miR-184 mimic significantly inhibited the tube formation of RF/6A in the presence and absence of VEGF induction, compared with the scrambled counterpart (Figs. 6A, 6B). On the other hand, the miR-184 inhibitor increased the tube formation of RF/6A in the presence and absence of VEGF induction, compared with the scrambled ones (Figs. 6C, 6D). 

Figure 6

 

MicroRNA-184 inhibited tube formation of RF/6A cells. A VEGF₁₆₅-induced tube formation model was applied in the RF/6A cells transfected with the miR-184 mimic or inhibitor for 48 hours. (A) Representative images of tube formation of RF/6A cells transfected with scramble or the mimic of miR-184 at 0 or 48 hours. Scale bar: 100 μm. (B) Statistical analysis of the data of tube formation of RF/6A cells transfected with scramble or the mimic of miR-184 after 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, ***P < 0.001.) (C) Representative images of tube formation of RF/6A cells transfected with scramble or the inhibitor of miR-184 at 0 or 48 hours. Scale bar: 100 μm. (D) Statistical analysis of the data of tube formation of RF/6A transfected with scramble or the inhibitor of miR-184 after 48 hours. (Data are presented as mean ± SEM, n = 3 in each group, *P < 0.05.)

Discussion

MicroRNA-184 is abundantly expressed in corneas, but its physiological and pathologic functions in this tissue are still largely unknown. We provide novel evidence that miR-184 down-regulation is associated with corneal neovascularization, whereas topical application of miR-184 can ameliorate corneal neovascularization, indicating that sufficient miR-184 in the cornea is important to maintain corneal avascularity and transparency under normal conditions. In addition, our results indicate that miR-184 plays a critical role in preventing corneal neovascularization through negatively regulating VEGF and the Wnt/β-catenin expression levels. This novel insight helps clarify some of the mechanisms underlying corneal neovascularization and reveals novel gene targets for treating corneal angiogenesis/neovascularization. 

Recently, multiple studies demonstrated that microRNAs, such as miR-126, the miR-17∼92 cluster, miR-378, miR-210, and miR-296, regulate angiogenesis in response to injury, developmental angiogenesis, and tumor angiogenesis.^5–8,20,21 It is also reported that the regulation of microRNAs is through manipulating the expression levels of proangiogenic and antiangiogenic factors and endothelial cell functions.²⁰ Little is known about the role of microRNAs in ocular neovascularization.⁶ Two indications of their involvement are that the miRNA cluster 23∼27∼24 elicits choroidal neovascularization control⁵ and that ischemia-induced neovascularization was associated with miR-184 down-regulation in the retina.¹⁴ Even though we provide here clear evidence of a regulatory role for miR-184 in a rat corneal suture model, future studies are needed using other animal models,²² along with microRNA microarray analysis and in situ miR-184 hybridization, to determine the specificity and universality of our finding. 

Vascular endothelial growth factor is a key proangiogenic factor and plays a vital role in the formation and development of vascularization.^23,24 Recently, several microRNAs, such as miR-195 and miR-503, were reported to regulate angiogenesis in tumor development.^17,19 In our previous study with the corneal suture model, we demonstrated that VEGF was significantly up-regulated.¹⁵ In our present investigation with the transfection of mimic and inhibitor of miR-184, we showed that miR-184 negatively regulated VEGF expression levels and suppressed VEGF-induced endothelial cell tube formation. Taken together, these findings suggest that miR-184 targets or modulates VEGF and that down-regulation of miR-184 may enhance corneal VEGF expression. 

Multiple microRNAs such as let-7f miRNA, miR-34, and miR-8 can activate or regulate the Wnt signaling pathway in stem cells and cancer cells.^25–27 A recent study revealed that miR-184 modulates canonical Wnt signaling through the regulation of frizzled-7 expression in the retina with ischemia-induced neovascularization.¹⁴ Here we demonstrated that corneal suture significantly increased the gene expression of frizzled-4, which is consistent with our earlier finding that corneal suture resulted in the activation of the Wnt signaling pathway.¹⁵ Furthermore, these results are also supported by our current report in which we show that miR-184 negatively regulates the key factor of Wnt pathway β-catenin with tranfection of mimic and inhibitor of miR-184 in both HUVECs and HCE cells. Our findings indicate that the Wnt/β-catenin signaling is regulated by miR-184, and Wnt/β-catenin is a target of miR-184 in the cornea. 

The biological roles of microRNAs are complicated. MicroRNAs can target or regulate many genes and multiple pathways. Although we provide novel evidence that corneal suture induces down-regulation of miR-184 and miR-814 might negatively regulate VEGF and Wnt/β-catenin signaling, additional investigations are needed to better delineate the mechanism of miR-184 in corneal neovascularization. For example, investigations should pinpoint other pathways or factors that miR-184 targets or regulates, clarify interactions between the pathways or factors that miR-184 targets or regulates, and look at the interplay between miR-184 and its potential targets or the regulation of miR-184 by other factors. 

Acknowledgments

The authors thank Peter Reinach, PhD, for the manuscript editing assistance. This work is supported by the Natural Science Foundation of China, Beijing, China (Grants 81400379 and 81170818) and the National High Technology Research and Development Program of China, Beijing, China (Grant 2012AA020507). The funders had no role in study design, data collection and analysis, decision regarding publishing, or preparation of the manuscript. 

Disclosure: R. Zong, None; T. Zhou, None; Z. Lin, None; X. Bao, None; Y. Xiu, None; Y. Chen, None; L. Chen, None; J.-X. Ma, None; Z. Liu, None; Y. Zhou, None