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Cornea  |   June 2015
MicroRNA Expression Profile and the Role of miR-204 in Corneal Wound Healing
Author Affiliations & Notes
  • Jianhong An
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Xiaoyan Chen
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Weiwei Chen
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Rongxin Liang
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Peter S. Reinach
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Dongsheng Yan
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • LiLi Tu
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Correspondence: LiLi Tu, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China; tulili@mail.eye.ac.cn. Dongsheng Yan, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China; dnaprotein@hotmail.com
  • Footnotes
     JA and XC contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3673-3683. doi:10.1167/iovs.15-16467
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      Jianhong An, Xiaoyan Chen, Weiwei Chen, Rongxin Liang, Peter S. Reinach, Dongsheng Yan, LiLi Tu; MicroRNA Expression Profile and the Role of miR-204 in Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2015;56(6):3673-3683. doi: 10.1167/iovs.15-16467.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: MicroRNAs (miRNAs) are endogenous short chain (∼22-nucleotide) noncoding RNAs that inhibit protein translation through binding to target mRNAs. Recent studies have implied that miRNAs play a regulatory role in corneal development. Here we profile their involvement in corneal epithelial renewal, develop an miRNA-target network that affects wound healing outcome, and investigate the function of miR-204 in this response.

Methods.: NanoString nCounter technology and bioinformatics analyzed miRNA expression levels and their targets during mouse corneal epithelial wound healing. Real-time RT-PCR was performed to detect miR-204 expression in mouse corneal epithelium. Human corneal epithelial cells (HCECs) were transfected with miR-204 using transfection reagent. MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) and a scratch wound-healing assay evaluated the effects of miR-204 expression on HCEC proliferation and migration, respectively. Cell cycle analysis was performed by flow cytometry. Expression of sirtuin 1 (SIRT1) was determined by Western blot analysis.

Results.: Fifteen miRNAs were dramatically downregulated, whereas 14 other miRNAs were markedly upregulated during corneal wound healing. Expression of miR-204 fell the most during this process. Transfection of miR-204 into HCECs led to a significant decline in cell proliferation and induced cell cycle G1-arrest. Furthermore, in these cells, miR-204 also inhibited migration. Sirtuin 1 was confirmed as a target of miR-204.

Conclusions.: During mouse corneal epithelial wound healing, a complex miRNA-gene network was resolved that is modulated by changes in miR-204 expression. Downregulation of this miRNA appears to be an essential response to injury since its decline promotes human corneal epithelial cell proliferation and migration. Therefore, miR-204 could be a biomarker of this process.

Corneal transparency and thinness maintenance are essential for normal vision. The preservation of these characteristics is in part dependent on the ability of the corneal epithelial layer to undergo continuous renewal. This renewal process replaces terminally differentiated cells that undergo senescence and prevents the epithelium from losing its barrier function against pathogenic infiltration into the underlying stromal layer. In addition, preserving barrier integrity helps offset the natural tendency of the stroma to imbibe excessive fluid, thicken, and thereby lose transparency.1,2 
Epithelial renewal depends on the ability of stem cells to proliferate and centripetally migrate from the corneal limbus and differentiate into basal layer proliferating cells. This regenerative process replaces the centrally located terminally differentiated cells undergoing senescence.3 A host of cytokines exert their influences through cognate receptor activation and linked signaling pathways, leading to time-dependent increases in gene expression modulating cell proliferation and migration as well as phenotypic expression. Recently, noncoding genes were described that are also expressed during corneal epithelial wound healing. Some of them act as switches to control the readout of genes eliciting increases in cell proliferation and migration. However, the characterization of the microRNA (miRNA) expression patterns affected by these responses to injury is incomplete and has been somewhat limited by reliance on microarray technology, which for one lacks sensitivity to identify changes in single corneas. 
MicroRNAs are small, 22-nucleotide, noncoding RNAs that posttranscriptionally regulate mRNA breakdown or elicit translational interference of tissue specific genes involved in the control of responses such as cell differentiation, proliferation, migration, cell death mechanisms, scarring, inflammation, and stem cell preservation.47 Such control by specific miRNAs can also be due to gene target degradation. Over the past decade, over 2000 miRNAs have been identified in humans. Yet with most of them, their functional roles have not yet been intensely characterized, especially in corneal development and diseases. Their contribution to such control is manifested through specific miRNAs.8 The identification of the involved miRNAs is of therapeutic importance because it reveals potential novel drug targets for hastening corneal epithelial wound healing in a clinical setting.911 
Some insight has been gained in identifying specific miRNAs that compromise corneal epithelial integrity in diabetes.9 One manifestation of diabetes can be increased corneal epithelial fragility and impaired epithelial wound healing subsequent to injury. These impairments are due to declines in cell proliferation, migration, and epithelial basement membrane attachment. They are reported to be associated with miR-146 and miR-424 upregulation, which leads to delays in epithelial wound closure and may contribute to increased epithelial fragility. On the other hand, in nondiabetic corneas, it has been suggested that miR-205 upregulation may be involved in stimulating the wound healing response. Such increases stimulate in human and mouse corneal epithelial cells proliferation and migration through inhibition of a 20 pS K+ channel designated KCNJ10-like.11 
Even though these two studies pinpoint several different miRNAs involved in the control of corneal epithelial wound healing, it is becoming increasingly apparent in some other tissues that there are a multitude of miRNAs with overlapping functions that can become dysregulated, resulting in a pathophysiological condition. As there is a paucity of corneal studies describing miRNA involvement in controlling cell proliferation and migration during wound healing, such insight is needed to identify novel procedures for hastening epithelial renewal that can be dysregulated in disease. 
We catalog here the differential effects on miRNA expression of corneal epithelial wounding in mice using products from NanoString Technologies, Inc. (Seattle, WA, USA). The analysis indicates that out of the 600 candidate miRNAs interrogated with this technique, only miR-184 and miR-204 levels were dramatically downregulated over 200-fold. Even though 14 other miRNAs were upregulated, miR-204 targeted genes appear to be essential for controlling cell proliferation and migration. This association is evident since miR-204 transfection into human corneal epithelial cells suppressed the increases in cell proliferation and migration induced by wounding these cultures. Bioinformatics network analysis identified a cohort of miRNA gene targets that elicit expression of mediators involved in the control of these responses as well as epithelial mesenchymal transition (EMT). We also confirmed SIRT1 as a target of miR-204. Our study indicates that miRNAs play important roles in cornea wound healing and miR-204 can be considered as a biomarker in this process. 
Materials and Methods
Corneal Wound Healing in the Mouse Eye Model in Vivo
Female 6- to 8-week-old C57BL/6 mice were anesthetized by combined intraperitoneal administration of xylazine (13 mg/kg) and ketamine (87 mg/kg), and then eyes were topically anesthetized with a drop of proparacaine hydrochloride eye drops (Alcon, Inc., Puurs, Belgium). The full thickness of the corneal epithelium up to the corneal/limbal border was removed using a commercial product with a 0.5-mm corneal rust ring remover (AlgerBrush II, The Alger Company, Inc., Lago Vista, TX, USA) under a stereomicroscope. Immediately after scraping, erythromycin eye ointment was applied to the wounded eyes to prevent bacterial infection. Healing was allowed to proceed until approximately only 10% of the wounded area remained, which took around 48 hours. Then the whole corneal epithelium was collected for RNA isolation from both the injured and the contralateral uninjured corneas. All animal treatments were performed in strict accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and approval of the Wenzhou Medical University Animal Care and Use Committee. 
NanoString nCounter miRNA Assay for miRNA Profiling
We used a mouse miRNA assay kit (nCounter, v1.3; NanoString Technologies, Inc.) for digital miRNA profiling in 100 ng total RNA extracted from each mouse whole corneal epithelium. Abundance levels of well-characterized and validated mouse (n = 578) and mouse-associated viral miRNAs (n = 33) were determined. The assay was performed according to the manufacturer's instructions. Data were normalized to the top 100 most highly expressed miRNAs in each sample, and the average +2 SD of six internal negative control probes were used to determine background detection threshold. To filter for expressed miRNAs, we computed the mean expression values of each miRNA across our cohort, and a threshold cutoff-point of 6.0 was selected based on background detection threshold. Values exceeding this cutoff-point were considered to be indicative of relevant miRNA expression. Accordingly, only those satisfying this criterion were taken as candidates for the following bioinformatics analysis. 
Bioinformatic and Statistical Analysis
Hierarchical Cluster Analysis.
Hierarchical clustering was performed with average linkage using open source clustering software (Cluster 3.0; Eisen Lab, University of California at Berkeley, CA, USA). The clustered heat map was visualized using interactive graphical software (TreeView; Eisen Lab, University of California at Berkeley). A limma algorithm was applied to filter the differentially expressed miRNAs under the different conditions.12 After performing the significance analysis and false discovery rate (FDR) analysis, we selected differentially expressed miRNAs using the following criteria: (1) P < 0.05, FDR <0.05; (2) fold change >2 or <0.05. Data were clustered based on Euclidian distance and each row was normalized using a z-score. 
Target Analysis.
We utilized TargetScan as a tool for predicting miRNA targets of the differentially expressed miRNAs. Next, we performed gene ontology (GO) analysis to facilitate elucidating the biological implications of unique genes in the significant or representative profiles of the differentially expressed miRNA gene targets in an experiment. We downloaded the GO annotations from the NCBI (http://www.ncbi.nlm.nih.gov/, in the public domain), UniProt (http://www.uniprot.org/, in the public domain), and Gene Ontology (http://www.geneontology.org/, in the public domain) sites. Fisher's exact test was applied to identify the significant GO categories and FDR was used to correct the P values. Pathway analysis was used to identify significant pathways linkage to differentially expressed genes according to KEGG database. We chose the Fisher's exact test to select the significant pathway, and the threshold of significance was defined based on its P value and FDR. 
Generation of miRNA-Target Networks.
We conducted the functional enrichment analysis of genes consisting of miRNA-target pairs using GO and pathway analyses results. Networks were generated by connecting gene nodes and differentially expressed miRNAs to represent direct and indirect biological relationships utilizing open source software (Cytoscape; Cytoscape Consortium, San Diego, CA, USA). 
Quantitative RT-PCR.
Total RNA was extracted from cornea tissues with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and its integrity was confirmed. We used 10 ng total RNA for cDNA synthesis by an miRNA kit (Taqman MicroRNA Reverse Transcription Kit; Applied Biosystems, Foster City, CA, USA). The expression level of miR-204 was quantified with the a commercial assay (Taqman MicroRNA Assay; Applied Biosystems), according to manufacturer's instructions. Real-time RT-PCR was performed using a real-time PCR system (7500 Fast Real-Time PCR System; Applied Biosystems). Expression of miR-204 was normalized to small nuclear U6 snRNA expression. 
Cell Proliferation Assay.
We cultured SV40-immortalized HCECs (gift from Araki Sasaki Kagoshima, Miyata Eye Clinic, Kagoshima, Japan), in supplemented Dulbecco's modified Eagle's medium (DMEM/F12; Invitrogen) containing 10% FBS (Invitrogen), as previously reported.13 We plated HCECs at 3 × 103 cells per well in 96-well plates (Costar, High Wycombe, UK) for each transfection. Transfections were performed using a transfection reagent (Lipofectamine RNAiMAX; Invitrogen). For each well, 50 nM miR-204 precursor molecule (Ambion, Austin, TX, USA) or a negative control (Ambion) was transfected. After 24-hour culture, cell proliferation was assessed using an assay kit (CellTiter 96 Aqueous; Promega Corp., Madison, WI, USA) according to manufacturer's instructions. 
Flow Cytometry Analysis.
Forty-eight hours after transfection with 50 nM miRNAs, HCECs (1 × 105) were stained with propidium iodide using a reagent kit (Cycle Test Plus DNA; Becton Dickinson, San Jose, CA, USA), then analyzed for DNA content with a flow cytometer (FACScaliber; Becton Dickinson). 
In Vitro Scratch Wound Assay.
Human corneal epithelial cells were grown in DMEM containing 10% FBS to approximately 60% confluence and transfected with either 50 nM miR-204 or a negative control. After 24 hours, the cell monolayers were scratched using a 100-μL pipette tip, washed twice with Hanks medium to remove the floating cells, then cultured in 2-mL fresh serum free medium. Photographs were taken immediately after scratching and at 12 hours after culture (Imager Z1; Zeiss, Jena, Germany). 
Western Blot Analysis.
Human corneal epithelial cells cells (1 × 105) were seeded and grown for 24 hours, transfected with miR-204 or negative control. Corneal wound healing in the mouse eye model was developed as described above. Western blot analysis was performed following the procedure as previously reported. Antibodies for SIRT1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Beverly, MA, USA). 
Results
miRNA Profile During Corneal Wound Healing
In six mice, corneal epithelial scraping was performed to monitor miRNA profile changes during wound healing. When the remaining wound area was approximately 10% of the initial debrided epithelial area, the tissue was harvested. The epithelial tissue was also removed from the uninjured control eye at this time. From each of the epithelial harvests, total RNA was extracted. Their miRNA expression profiles were individually analyzed by NanoString nCounter technology. As anticipated, there was substantial variation in the low-expressed miRNAs in each of the controls (Fig. 1A) and during epithelial healing (Fig. 1B). Therefore, for obtaining RNA comparisons across groups or performing association analyses by subject characteristics, it was important to consider only relevantly expressed miRNAs. Based on the distribution of mean miRNA levels in the entire cohort, an expression threshold of 6.0 for the log2 miRNA probe count was selected (Fig. 1). The 30 most highly expressed corneal miRNAs derived from the six normal corneal epithelial samples are listed in Table 1, and Supplementary Table S1 contains the entire list of identifiable miRNAs, their mean levels of expression, standard deviations, and coefficient of variation as well as a comparison between the control and wounded groups. 
Figure 1
 
Changes in corneal epithelial miRNA expression following wounding. The lowest levels of miRNAs show the greatest variation in both control corneal epithelia (A) and repaired injury corneal epithelia (B). Each point represents one measured feature in the miRNA assay. x-axes indicate mean abundance (log2) and y-axes indicate coefficient of variation. The dashed red line indicates the threshold cutoff and the blue dots represent miRNAs abandoned.
Figure 1
 
Changes in corneal epithelial miRNA expression following wounding. The lowest levels of miRNAs show the greatest variation in both control corneal epithelia (A) and repaired injury corneal epithelia (B). Each point represents one measured feature in the miRNA assay. x-axes indicate mean abundance (log2) and y-axes indicate coefficient of variation. The dashed red line indicates the threshold cutoff and the blue dots represent miRNAs abandoned.
Table 1
 
Thirty Most Commonly Expressed miRNAs in Mouse Corneal Epithelia
Table 1
 
Thirty Most Commonly Expressed miRNAs in Mouse Corneal Epithelia
The most differentially expressed miRNAs were hierarchically clustered and displayed in a heat map (Fig. 2). All the differentially expressed miRNAs are listed in Table 2. As indicated, 15 miRNAs were remarkably downregulated during the corneal wound healing process, of which miR-204 and miR-184 were the most downregulated miRNAs (their values of log2fold change are −8.06 and −5.99, corresponding to declines of 267-fold and 64-fold, respectively). In contrast, 14 other miRNAs were instead upregulated in corneal wound healing. The numbers of miRNAs upregulated or downregulated were similar, however, many miRNAs in the most highly expressed group were downregulated more than any of the other identifiable miRNAs during wound healing. 
Figure 2
 
Hierarchical clustering analysis of differentially expressed miRNAs during corneal epithelial wound healing. The mouse corneal wound healing model involved epithelial debridement, and the miRNA expression profile in corneal epithelium during corneal wound healing process was analyzed by NanoString nCounter technology.
Figure 2
 
Hierarchical clustering analysis of differentially expressed miRNAs during corneal epithelial wound healing. The mouse corneal wound healing model involved epithelial debridement, and the miRNA expression profile in corneal epithelium during corneal wound healing process was analyzed by NanoString nCounter technology.
Table 2
 
Differentially Expressed miRNAs in Mouse Corneal Epithelial Wound Healing in Comparison With Their Uninjured Control
Table 2
 
Differentially Expressed miRNAs in Mouse Corneal Epithelial Wound Healing in Comparison With Their Uninjured Control
Bioinformatic Analyses Reveal miRNA-Target Network During Corneal Wound Healing
To identify the genes that mediate control of mouse corneal re-epithelialization, such as cell proliferation, wound healing, and cell-cycle progression, we conducted GO enrichment analysis. This was done by using these differentially expressed miRNAs as well as a set of their significant predicted target genes involved in cell proliferation, cell cycle and wound healing for resolving a miRNA-target network (Fig. 3). This approach affirms that corneal epithelial cells share core survival pathways as well as different regulatory patterns of miRNAs expression during wound healing. 
Figure 3
 
Network of differentially expressed miRNAs with their targets in mouse corneal re-epithelialization during wound healing. Squares represent differentially upregulated miRNAs (red) or downregulated miRNAs (green), while nodes represent their putative targets and links represent the regulation of miRNAs on their target genes. The background color behind the clusters represents a cluster's function in relation to one of the re-epithelialization hallmarks: wound healing (cyan), cell cycle (blue), and cell proliferation (orange). Cytoscape was used to present and visualize the networks.
Figure 3
 
Network of differentially expressed miRNAs with their targets in mouse corneal re-epithelialization during wound healing. Squares represent differentially upregulated miRNAs (red) or downregulated miRNAs (green), while nodes represent their putative targets and links represent the regulation of miRNAs on their target genes. The background color behind the clusters represents a cluster's function in relation to one of the re-epithelialization hallmarks: wound healing (cyan), cell cycle (blue), and cell proliferation (orange). Cytoscape was used to present and visualize the networks.
To simplify the analysis, only EMT related genes were included, which are also predicted targets of these differentially expressed miRNAs as well as those of miR-204 or 184 (Fig. 4). Thirteen potential target genes of miR-204 and one potential target gene of miR-184 are associated with EMT. These genes include markers of EMT such as CDH2, transcription factors, ZEB1, and inducers of EMT such as ESR1, which are known to contribute to the induction of EMT, members of VEGF, PI3K-Akt, Wnt, and TGF-beta signaling pathways such as PIK3CB, SOS1, TCF7L1, SP1, and TGFBR2, which are involved in induction of EMT as well as wound healing. 
Figure 4
 
MicroRNA-target network. The relationship of the miRNAs and their predicted genes were counted by their differential expression values, and according to the interactions of miRNAs and genes in the Sanger microRNA database to build the MicroRNA-gene network. Green: Downregulated miRNAs. Red: Upregulated miRNAs. Silver: Targets of miR-204 and 184. Blue: EMT-related targets. Violet: EMT-related targets of miR-184 and miR-204.
Figure 4
 
MicroRNA-target network. The relationship of the miRNAs and their predicted genes were counted by their differential expression values, and according to the interactions of miRNAs and genes in the Sanger microRNA database to build the MicroRNA-gene network. Green: Downregulated miRNAs. Red: Upregulated miRNAs. Silver: Targets of miR-204 and 184. Blue: EMT-related targets. Violet: EMT-related targets of miR-184 and miR-204.
Epithelial mesenchymal transition in wound healing is induced through the activation of an intricate network of multiple signaling pathways, including TGF-beta, Wnt, MAPK, VEGF, Notch, and PI3K-Akt pathways.14 To better delineate the signaling pathways involved in corneal epithelial cells during wound healing, we conducted a network analysis of mouse miR-204 and its putative targets. Our approach utilized gene ontology analysis and pathway analysis toward the predicted target genes of miR-204 (Fig. 5). Many genes of TGF-beta, Wnt, MAPK, VEGF, mTOR, ErbB, and PI3K-Akt signaling pathway intermediates are potential targets of miR-204, which is downregulated more than 200-fold during corneal epithelial cell wound healing. This prediction is supportive of earlier studies showing that these signaling pathways play important roles in mediating corneal epithelial cell layer wound healing. In addition, genes that are potential miR-204 targets were identified that are involved in cytoskeleton formation, cell migration, cell cycle progression, and cell proliferation. This agreement validates their described roles in corneal epithelial wound healing. 
Figure 5
 
A network of mouse miR-204 and its putative targets. Genes in several GO-term and pathway-term were selected to construct this network, which are based on the GO and pathway analysis toward the predicted target genes of mouse miR-204.
Figure 5
 
A network of mouse miR-204 and its putative targets. Genes in several GO-term and pathway-term were selected to construct this network, which are based on the GO and pathway analysis toward the predicted target genes of mouse miR-204.
miR-204 Is Dramatically Decreased in the Corneal Epithelial Wound Healing Process
To confirm the over 200-fold miR-204 downregulation that occurred during corneal wound healing—which was identified using NanoString nCounter technology—real-time RT-PCR was performed. In six different mouse corneal epithelial cell layers undergoing wound healing, miR-204 expression also dramatically declined compared with its level in tissue isolated from control uninjured corneas (Fig. 6). 
Figure 6
 
miR-204 is dramatically downregulated during corneal epithelial wound healing process. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of miR-204 in corneal epithelium during wound healing process was examined by real-time RT-PCR. We used U6 snRNA as an internal control. The numbers on the ordinate identify epithelial samples each obtained from different mice.
Figure 6
 
miR-204 is dramatically downregulated during corneal epithelial wound healing process. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of miR-204 in corneal epithelium during wound healing process was examined by real-time RT-PCR. We used U6 snRNA as an internal control. The numbers on the ordinate identify epithelial samples each obtained from different mice.
miR-204 Inhibits HCEC Cell Proliferation and Cell Cycle Progression
Since miR-204 expression was nearly undetectable during corneal wound healing, we then sought to determine whether increasing miR-204 expression instead suppressed this regenerative response. These cells were transfected with either the miR-204 precursor or a negative control. After transfection, the MTS assay was carried out to assess proliferation inhibition after 48 hours. Compared with that in the control (Fig. 7A), miR-204 caused a dramatic inhibition of cell proliferation. Consistent with the finding that miR-204 transfection inhibited cell proliferation, their percentage in G1 cell cycle arrest was 62%, whereas this value fell to 40% in those cells transfected instead with the negative control (Fig. 7B). 
Figure 7
 
Effect of miR-204 on the proliferation of human corneal epithelial cells. (A) We performed an MTS cell proliferation assay 48 hours after transfection with either a miR-204 molecule or a negative control (NC) that does not encode for any known miRNA. The number of cells transfected with miR-204 was significantly decreased than that of cells transfected with negative control. *Differences in cell proliferation between miR-204 and NC transfected cells were significant (P < 0.01). (B) Ectopic miR-204 induces human corneal epithelial cell cycle G1 arrest. Human corneal epithelial cells were collected 48 hours after transfection with miR-204 or NC, stained with propidium iodide and analyzed by flow cytometry. Ten thousand cells were evaluated in each sample. Representative results are depicted.
Figure 7
 
Effect of miR-204 on the proliferation of human corneal epithelial cells. (A) We performed an MTS cell proliferation assay 48 hours after transfection with either a miR-204 molecule or a negative control (NC) that does not encode for any known miRNA. The number of cells transfected with miR-204 was significantly decreased than that of cells transfected with negative control. *Differences in cell proliferation between miR-204 and NC transfected cells were significant (P < 0.01). (B) Ectopic miR-204 induces human corneal epithelial cell cycle G1 arrest. Human corneal epithelial cells were collected 48 hours after transfection with miR-204 or NC, stained with propidium iodide and analyzed by flow cytometry. Ten thousand cells were evaluated in each sample. Representative results are depicted.
miR-204 Transfection Inhibits HCEC Migration
To assess if miR-204 upregulation also inhibits wound closure through suppression of cell migration, the scratch wound assay was carried out to make this determination. Twelve hours after injury, it is apparent that transfected miR-204 cells migrated slower than those transfected with an irrelevant sequence since the remaining open wound area was larger in the transfected miR-204 cells than in their negative control (Fig. 8). 
Figure 8
 
miR-204 inhibits human corneal epithelial cell migration. Human corneal epithelial cells were transfected with miR-204 or NC for 24 hours and in vitro wound-healing assay was conducted. Scratches were made in the cultured human corneal epithelial monolayer. Then the cells were cultured for 0, 12 hours in DMEM without serum. The scratched regions were photographed using a phase-contrast microscope (×100). Representative results are presented.
Figure 8
 
miR-204 inhibits human corneal epithelial cell migration. Human corneal epithelial cells were transfected with miR-204 or NC for 24 hours and in vitro wound-healing assay was conducted. Scratches were made in the cultured human corneal epithelial monolayer. Then the cells were cultured for 0, 12 hours in DMEM without serum. The scratched regions were photographed using a phase-contrast microscope (×100). Representative results are presented.
SIRT1 Is a Target of miR-204
To further determine the validity of our bioinformatic analyses of the miRNA target network, we selected SIRT1 gene as a candidate for validation. We used Western blotting to detect SIRT1 expression in HCECs after being transfected with miR-204 or negative control. Transfection of miR-204 resulted in a remarkable decrease in SIRT1 expression (Fig. 9A). We then further determined whether SIRT1 expression varies during mouse corneal wound healing. As shown in Figure 9B, the level of SIRT1 was dramatically upregulated in three different corneal epithelial layers undergoing wound healing, compared with its level in tissue isolated from control uninjured corneas. Taken together, our results demonstrate that SIRT1 is a bona fide target of miR-204. 
Figure 9
 
Sirtuin 1 is target of miR-204. (A) miR-204 downregulated SIRT1 expression in HCECs; HCECs were transfected with miR-204 or NC, then used for Western blot analysis with SIRT1 antibody. GAPDH was used as an internal control. (B) Expression of SIRT1 is elevated during mouse corneal wound healing. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of SIRT1 in corneal epithelium during wound healing process was measured by Western blot analysis.
Figure 9
 
Sirtuin 1 is target of miR-204. (A) miR-204 downregulated SIRT1 expression in HCECs; HCECs were transfected with miR-204 or NC, then used for Western blot analysis with SIRT1 antibody. GAPDH was used as an internal control. (B) Expression of SIRT1 is elevated during mouse corneal wound healing. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of SIRT1 in corneal epithelium during wound healing process was measured by Western blot analysis.
Discussion
Emerging evidence suggests that miRNAs have a regulatory role in corneal development and pathology. Dicer, a pivotal ribonuclease in miRNA processing, plays an essential role in eye development. Targeted deletion of Dicer results in microphthalmia and thinner corneal epithelium, which implicates the importance of miRNAs in ocular and corneal development.15 There is more direct evidence indicating the importance of miRNAs in corneal epithelial development and physiology. For example, miR-31 modulation affects corneal epithelial glycogen metabolism and keratinocyte differentiation.16,17 Similarly, changes in miR-184 and miR-205 expression alter SHIP2 levels in epithelia.18 Recently, miRNAs have been linked to the corneal epithelial wound healing process. In one study, a miRNA signature was identified during wound healing using miRNA PCR array; miR-133b decreased more than others by 48%, which was associated with declines in the expression of genes mediating stromal fibrosis.10 Even though these studies are indeed informative, their resolving power and sensitivity is somewhat limited. 
In the present study, we adopted a novel biotechnology approach, namely NanoString nCounter, which resolves endogenous miRNAs without amplification and allows comparisons of miRNA expression profiles within and between individual samples. The nCounter platform is an efficient multiplex system using digital technology to rapidly count individual RNA molecules in a single reaction without performing first mRNA to cDNA conversion or amplification. It is a very reliable high throughput assay having greater sensitivity and reproducibility than microarray measurements.19,20 This system is as sensitive and precise as real-time TaqMan RT-PCR.21 By using target-specific color-coded probe pairs in conjunction with a single molecule imaging system, it resolves endogenous mRNA targets with a precision and sensitivity of less than one transcript copy per cell. Similar to RNA-seq, this advanced platform uses discrete count measurements, while its sensitivity is sufficient to resolve variations in miRNA expression profiles between individual epithelial cell samples from different mice. With this capability, it is possible to identify with greater accuracy pattern differences occurring during wound healing. 
We identified a specific miRNA signature in corneal epithelial wound healing, namely, declines in miR-204 and miR-184 were greater than those of all other candidates. This miRNA signature is distinct from a previous study, mainly due to their use of a different injury model. Our injury-induced wound healing model is strictly limited to the corneal epithelium, while the other model “removed all of the corneal epithelial cell layers and some of the stroma to simulate photorefractive keratectomy (PRK).”10 In addition, our use of the NanoString platform for miRNA analysis may be another reason why the PRK-simulation study failed to identify the larger declines we saw in miR-184 and miR-204 expression levels. We identified 112 miRNAs out of which 29 were differentially expressed during corneal epithelial wound healing. In normal corneal epithelia, we resolved the breadth as well as the abundance of miRNA expression. The top three most abundant miRNAs are miR-184, miR-204, and miR-205, consistent with a previous report.18 In one study, miR-205 expression was shown to facilitate the wound healing process in HCECs by suppressing 20 pS K+ channel activity.11 It is unclear if such a decline is actually related to an increase in cell proliferation. In rabbit corneal epithelial cells, their mitogenic response to epidermal growth factor (EGF) receptor activation by EGF is associated with increases in such channel activity.22 Channel activation of K+ in turn causes membrane voltage hyperpolarization, which contributes to increasing Ca2+ influx required for activating downstream signaling events mediating mitogenic responses to EGF receptor activation.23,24 However, in our study, miR-205 expression was unchanged. 
We found miR-204 is highly expressed in various eye tissues, such as the corneal epithelium, lens epithelium, retina and RPE.25,26 Using medaka fish as a vertebrate model, miR-204 is essential in lens and retinal development.27 Furthermore, miR-204 is critical in human RPE cell physiology and epithelial polarization.26 Despite being identified as one of the most abundant miRNAs in the corneal epithelium, the role of miR-204 was elusive in the cornea. Combined with NanoString nCounter technology and real-time RT-PCR, we definitively demonstrated a dramatic downregulation of miR-204 during corneal epithelial wound healing. Functional assays further indicated that increasing miR-204 expression levels toward those in HCEC led to retardation of cell proliferation and migration. Therefore, downregulation of miR-204 appears to sizably contribute to hastening epithelial wound healing subsequent to injury. 
During corneal epithelial wound healing, the remaining progenitor corneal epithelial cells at the periphery may undergo EMT as they migrate over the de-epithelialized area, which is associated with increases in cell proliferation and migration and loss of epithelial cell polarity. These changes are elicited by increases in epithelial cell TGF-beta expression, which in turn induces SMAD2/3/4, p38, and ERK1/2MAPK signaling transcription factor expression, ultimately leading to cell cycle progression and migratory activity.28,29 Our reported miR-204 expression decline is consistent with another study showing that miR-204 downregulation during EMT led to human posterior capsule opacification.30 Therefore, during re-epithelialization, miR-204 acts as an EMT suppressor since its downregulation accelerates cell migration. Although the underlying molecular mechanisms controlling EMT activation remain to be elucidated, a few miR-204 target genes have been verified, including SMAD4, SLUG, and SIRT1.3032 We also identified hundreds of putative gene targets of miR-204 and miR-184 in silico analysis, and developed a complicated miRNA-target network in wound healing. Specifically, we have demonstrated that SIRT1 is a bona fide gene target of miR-204 both in HCECs and mouse corneal wound healing by Western blot analysis. In a previous study, SIRT1 has been proven to enhance cell proliferation or migration in corneal wound healing.33 These results implicate miR-204 and its target SIRT1 in corneal wound healing. Interestingly, SMAD4 is a target of miR-204 in human lens epithelial cells,30 but it is not a predicted target of miR-204 in mice (Fig. 4), which may suggest that there is a different type of epigenetic control in humans than in mice. Although the accuracy of this suggestion requires future experimental verification, it nonetheless provides valuable clues for further investigation into the molecular mechanisms underlying miRNA regulation of epithelial wound healing. Future studies will focus on deciphering the precise regulatory mechanisms of miR-204 and miR-184. 
MicroRNAs can regulate multiple target genes and thereby modulate the expression of their gene products in disease mediating signaling pathways. This possibility makes it problematic in such cases to develop therapeutic options by merely targeting single miRNAs. However, in some cases, targeting specific miRNAs is a proven promising therapeutic option. For example, an antisense oligonucleotide blocking miR-122 ameliorates HCV (hepatitis C virus)–induced liver damage in primates,34 and has a potent anti-HCV effect in clinical trials.35 So targeting a specific miRNA, like miR-204, warrants further evaluation for promoting epithelial wound healing. 
In summary, we identified a specific miRNA expression profile of corneal epithelial wound healing in mice using NanoString technology. The analysis identifies 29 differentially expressed miRNAs, including 15 downregulated and 14 upregulated miRNAs. Out of 15 that underwent this change, miR-204 and miR-184 are the most dramatically downregulated miRNAs. Despite some of the miRNAs undergoing upregulation, miR-204 targeted genes appear to be essential for controlling cell proliferation and migration. This association is evident since miR-204 transfection into human corneal epithelial cells suppressed the increases in cell proliferation and migration induced by wounding these cultures. Bioinformatics network analysis identified a cohort of miRNA gene targets that elicit expression of mediators involved in the control of these responses as well as EMT. Our study indicates that miRNAs play important roles in corneal epithelial wound healing and miR-204 can be considered as a novel biomarker and as a potential target for optimizing this process in a clinical setting. 
Acknowledgments
We thank Bo Zhang and Jie Zong (Novel Bioinformatics Ltd., Co., Shanghai, China) for their technical assistance in the bioinformatic analysis. 
Supported in part by the National Natural Science Foundation of China Grant 81170821, 973 Projects (2011CB504605 & 2012CB722303) from the Ministry of Science and Technology of China, and Science Foundation of Wenzhou Medical University QTJ11020. 
Disclosure: J. An, None; X. Chen, None; W. Chen, None; R. Liang, None; P.S. Reinach, None; D. Yan, None; L. Tu, None 
References
Qazi Y, Wong G, Monson B, Stringham J, Ambati BK. Corneal transparency: genesis, maintenance and dysfunction. Brain Res Bull. 2010; 81: 198–210.
Lu L, Reinach PS, Kao WW. Corneal epithelial wound healing. Exp Biol Med (Maywood). 2001; 226: 653–664.
Schlotzer-Schrehardt U, Kruse FE. Identification and characterization of limbal stem cells. Exp Eye Res. 2005; 81: 247–264.
Carrington JC, Ambros V. Role of microRNAs in plant and animal development. Science. 2003; 301: 336–338.
Bartel DP. MicroRNAs: genomics biogenesis, mechanism, and function. Cell. 2004; 116: 281–297.
Foshay KM, Gallicano GI. Small RNAs big potential: the role of microRNAs in stem cell function. Curr Stem Cell Res Ther. 2007; 2: 264–271.
He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004; 5: 522–531.
Karali M, Peluso I, Gennarino VA et al. miRNeye: a microRNA expression atlas of the mouse eye. BMC Genomics. 2010; 11: 715.
Funari VA, Winkler M, Brown J, Dimitrijevich SD, Ljubimov AV, Saghizadeh M. Differentially expressed wound healing-related microRNAs in the human diabetic cornea. PLoS One. 2013; 8: e84425.
Robinson PM, Chuang TD, Sriram S et al. MicroRNA signature in wound healing following excimer laser ablation: role of miR-133b on TGFbeta1, CTGF, SMA, and COL1A1 expression levels in rabbit corneal fibroblasts. Invest Ophthalmol Vis Sci. 2013; 54: 6944–6951.
Lin D, Halilovic A, Yue P, et al. Inhibition of miR-205 impairs the wound-healing process in human corneal epithelial cells by targeting KIR4.1 (KCNJ10). Invest Ophthalmol Vis Sci. 2013; 54: 6167–6178.
Wettenhall JM Smyth GK. limmaGUI: a graphical user interface for linear modeling of microarray data. Bioinformatics. 2004; 20: 3705–3706.
Pan Z, Wang Z, Yang H, Zhang F, Reinach PS. TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells. Invest Ophthalmol Vis Sci. 2011; 52: 485–493.
Tam WL, Weinberg RA. The epigenetics of epithelial-mesenchymal plasticity in cancer. Nat Med. 2013; 19: 1438–1449.
Li Y, Piatigorsky J. Targeted deletion of Dicer disrupts lens morphogenesis, corneal epithelium stratification, and whole eye development. Dev Dyn. 2009; 238: 2388–2400.
Peng H, Hamanaka RB, Katsnelson J et al. MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism. FASEB J. 2012; 26: 3140–3147.
Peng H, Kaplan N, Hamanaka RB, et al. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation. Proc Natl Acad Sci U S A. 2012; 109: 14030–14034.
Yu J, Ryan DG, Getsios S, Oliveira-Fernandes M, Fatima A, Lavker RM. MicroRNA-184 antagonizes microRNA-205 to maintain SHIP2 levels in epithelia. Proc Natl Acad Sci U S A. 2008; 105: 19300–19305.
Geiss GK, Bumgarner RE, Birditt B et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol. 2008; 26: 317–325.
Mestdagh P, Hartmann N, Baeriswyl L, et al. Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Nat Methods. 2014; 11: 809–815.
Kulkarni MM. Digital multiplexed gene expression analysis using the NanoString nCounter system. Curr Protoc Mol Biol. 2011; Chapter 25:Unit25B.10.
Roderick C, Reinach PS, Wang L, Lu L. Modulation of rabbit corneal epithelial cell proliferation by growth factor-regulated K(+) channel activity. J Membr Biol. 2003; 196: 41–50.
Yang H, Sun X, Wang Z et al. EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells. J Membr Biol. 2003; 194: 47–58.
Wang Z, Bildin VN, Yang H, Capo-Aponte JE, Yang Y, Reinach PS. Dependence of corneal epithelial cell proliferation on modulation of interactions between ERK1/2 and NKCC1. Cell Physiol Biochem. 2011; 28: 703–714.
Ryan DG, Oliveira-Fernandes M, Lavker RM. MicroRNAs of the mammalian eye display distinct and overlapping tissue specificity. Mol Vis. 2006; 12: 1175–1184.
Wang FE, Zhang C, Maminishkis A et al. MicroRNA-204/211 alters epithelial physiology. FASEB J. 2010; 24: 1552–1571.
Conte I, Carrella S, Avellino R, et al. miR-204 is required for lens and retinal development via Meis2 targeting. Proc Natl Acad Sci U S A. 2010; 107: 15491–15496.
Aomatsu K, Arao T, Sugioka K et al. TGF-beta induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways in a human corneal epithelial cell line. Invest Ophthalmol Vis Sci. 2011; 52: 2437–2443.
Aomatsu K, Arao T, Abe K, et al. Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells. Invest Ophthalmol Vis Sci. 2012; 53: 751–756.
Wang Y, Li W, Zang X et al. MicroRNA-204-5p regulates epithelial-to-mesenchymal transition during human posterior capsule opacification by targeting SMAD4. Invest Ophthalmol Vis Sci. 2013; 54: 323–332.
Qiu YH, Wei YP, Shen NJ, et al. miR-204 inhibits epithelial to mesenchymal transition by targeting slug in intrahepatic cholangiocarcinoma cells. Cell Physiol Biochem. 2013; 32: 1331–1341.
Zhang L, Wang X, Chen P. MiR-204 down regulates SIRT1 and reverts SIRT1-induced epithelial-mesenchymal transition anoikis resistance and invasion in gastric cancer cells. BMC Cancer. 2013; 13: 290.
Wang Y, Zhao X, Shi D, et al. Overexpression of SIRT1 promotes high glucose-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3/IGF-1R/AKT pathway. Invest Ophthalmol Vis Sci. 2013; 54: 3806–3814.
Lanford RE, Hildebrandt-Eriksen ES, Petri A et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science. 2010; 327: 198–201.
Janssen HL, Reesink HW, Lawitz EJ, et al. Treatment of HCV infection by targeting microRNA. New Engl J Med. 2013; 368: 1685–1694.
Figure 1
 
Changes in corneal epithelial miRNA expression following wounding. The lowest levels of miRNAs show the greatest variation in both control corneal epithelia (A) and repaired injury corneal epithelia (B). Each point represents one measured feature in the miRNA assay. x-axes indicate mean abundance (log2) and y-axes indicate coefficient of variation. The dashed red line indicates the threshold cutoff and the blue dots represent miRNAs abandoned.
Figure 1
 
Changes in corneal epithelial miRNA expression following wounding. The lowest levels of miRNAs show the greatest variation in both control corneal epithelia (A) and repaired injury corneal epithelia (B). Each point represents one measured feature in the miRNA assay. x-axes indicate mean abundance (log2) and y-axes indicate coefficient of variation. The dashed red line indicates the threshold cutoff and the blue dots represent miRNAs abandoned.
Figure 2
 
Hierarchical clustering analysis of differentially expressed miRNAs during corneal epithelial wound healing. The mouse corneal wound healing model involved epithelial debridement, and the miRNA expression profile in corneal epithelium during corneal wound healing process was analyzed by NanoString nCounter technology.
Figure 2
 
Hierarchical clustering analysis of differentially expressed miRNAs during corneal epithelial wound healing. The mouse corneal wound healing model involved epithelial debridement, and the miRNA expression profile in corneal epithelium during corneal wound healing process was analyzed by NanoString nCounter technology.
Figure 3
 
Network of differentially expressed miRNAs with their targets in mouse corneal re-epithelialization during wound healing. Squares represent differentially upregulated miRNAs (red) or downregulated miRNAs (green), while nodes represent their putative targets and links represent the regulation of miRNAs on their target genes. The background color behind the clusters represents a cluster's function in relation to one of the re-epithelialization hallmarks: wound healing (cyan), cell cycle (blue), and cell proliferation (orange). Cytoscape was used to present and visualize the networks.
Figure 3
 
Network of differentially expressed miRNAs with their targets in mouse corneal re-epithelialization during wound healing. Squares represent differentially upregulated miRNAs (red) or downregulated miRNAs (green), while nodes represent their putative targets and links represent the regulation of miRNAs on their target genes. The background color behind the clusters represents a cluster's function in relation to one of the re-epithelialization hallmarks: wound healing (cyan), cell cycle (blue), and cell proliferation (orange). Cytoscape was used to present and visualize the networks.
Figure 4
 
MicroRNA-target network. The relationship of the miRNAs and their predicted genes were counted by their differential expression values, and according to the interactions of miRNAs and genes in the Sanger microRNA database to build the MicroRNA-gene network. Green: Downregulated miRNAs. Red: Upregulated miRNAs. Silver: Targets of miR-204 and 184. Blue: EMT-related targets. Violet: EMT-related targets of miR-184 and miR-204.
Figure 4
 
MicroRNA-target network. The relationship of the miRNAs and their predicted genes were counted by their differential expression values, and according to the interactions of miRNAs and genes in the Sanger microRNA database to build the MicroRNA-gene network. Green: Downregulated miRNAs. Red: Upregulated miRNAs. Silver: Targets of miR-204 and 184. Blue: EMT-related targets. Violet: EMT-related targets of miR-184 and miR-204.
Figure 5
 
A network of mouse miR-204 and its putative targets. Genes in several GO-term and pathway-term were selected to construct this network, which are based on the GO and pathway analysis toward the predicted target genes of mouse miR-204.
Figure 5
 
A network of mouse miR-204 and its putative targets. Genes in several GO-term and pathway-term were selected to construct this network, which are based on the GO and pathway analysis toward the predicted target genes of mouse miR-204.
Figure 6
 
miR-204 is dramatically downregulated during corneal epithelial wound healing process. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of miR-204 in corneal epithelium during wound healing process was examined by real-time RT-PCR. We used U6 snRNA as an internal control. The numbers on the ordinate identify epithelial samples each obtained from different mice.
Figure 6
 
miR-204 is dramatically downregulated during corneal epithelial wound healing process. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of miR-204 in corneal epithelium during wound healing process was examined by real-time RT-PCR. We used U6 snRNA as an internal control. The numbers on the ordinate identify epithelial samples each obtained from different mice.
Figure 7
 
Effect of miR-204 on the proliferation of human corneal epithelial cells. (A) We performed an MTS cell proliferation assay 48 hours after transfection with either a miR-204 molecule or a negative control (NC) that does not encode for any known miRNA. The number of cells transfected with miR-204 was significantly decreased than that of cells transfected with negative control. *Differences in cell proliferation between miR-204 and NC transfected cells were significant (P < 0.01). (B) Ectopic miR-204 induces human corneal epithelial cell cycle G1 arrest. Human corneal epithelial cells were collected 48 hours after transfection with miR-204 or NC, stained with propidium iodide and analyzed by flow cytometry. Ten thousand cells were evaluated in each sample. Representative results are depicted.
Figure 7
 
Effect of miR-204 on the proliferation of human corneal epithelial cells. (A) We performed an MTS cell proliferation assay 48 hours after transfection with either a miR-204 molecule or a negative control (NC) that does not encode for any known miRNA. The number of cells transfected with miR-204 was significantly decreased than that of cells transfected with negative control. *Differences in cell proliferation between miR-204 and NC transfected cells were significant (P < 0.01). (B) Ectopic miR-204 induces human corneal epithelial cell cycle G1 arrest. Human corneal epithelial cells were collected 48 hours after transfection with miR-204 or NC, stained with propidium iodide and analyzed by flow cytometry. Ten thousand cells were evaluated in each sample. Representative results are depicted.
Figure 8
 
miR-204 inhibits human corneal epithelial cell migration. Human corneal epithelial cells were transfected with miR-204 or NC for 24 hours and in vitro wound-healing assay was conducted. Scratches were made in the cultured human corneal epithelial monolayer. Then the cells were cultured for 0, 12 hours in DMEM without serum. The scratched regions were photographed using a phase-contrast microscope (×100). Representative results are presented.
Figure 8
 
miR-204 inhibits human corneal epithelial cell migration. Human corneal epithelial cells were transfected with miR-204 or NC for 24 hours and in vitro wound-healing assay was conducted. Scratches were made in the cultured human corneal epithelial monolayer. Then the cells were cultured for 0, 12 hours in DMEM without serum. The scratched regions were photographed using a phase-contrast microscope (×100). Representative results are presented.
Figure 9
 
Sirtuin 1 is target of miR-204. (A) miR-204 downregulated SIRT1 expression in HCECs; HCECs were transfected with miR-204 or NC, then used for Western blot analysis with SIRT1 antibody. GAPDH was used as an internal control. (B) Expression of SIRT1 is elevated during mouse corneal wound healing. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of SIRT1 in corneal epithelium during wound healing process was measured by Western blot analysis.
Figure 9
 
Sirtuin 1 is target of miR-204. (A) miR-204 downregulated SIRT1 expression in HCECs; HCECs were transfected with miR-204 or NC, then used for Western blot analysis with SIRT1 antibody. GAPDH was used as an internal control. (B) Expression of SIRT1 is elevated during mouse corneal wound healing. The mouse corneal wound healing model used epithelial scrape injury method. The expression level of SIRT1 in corneal epithelium during wound healing process was measured by Western blot analysis.
Table 1
 
Thirty Most Commonly Expressed miRNAs in Mouse Corneal Epithelia
Table 1
 
Thirty Most Commonly Expressed miRNAs in Mouse Corneal Epithelia
Table 2
 
Differentially Expressed miRNAs in Mouse Corneal Epithelial Wound Healing in Comparison With Their Uninjured Control
Table 2
 
Differentially Expressed miRNAs in Mouse Corneal Epithelial Wound Healing in Comparison With Their Uninjured Control
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