MicroRNA-204-5p–Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas

Abstract

Purpose.: We investigated how the microRNA (miRNA) modifies the expression of silent mating type information regulation 2 homolog 1 (SIRT1) in diabetic corneas.

Methods.: The bioinformatic assay was used to predict which miRNAs might regulate the expression of SIRT1. A lipid transfection protocol was used to upregulate or knockdown the miRNA expression in TKE2 cells. Adenovirus-expressing short interfering RNA was used to knockdown the expression of SIRT1 in TKE2 cells and Ins2^Akita/+ mice were used to evaluate how miRNA promotes diabetic corneal epithelial wound healing. Cell cycle status was determined by flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the cell viability.

Results.: Nine miRNAs were selected for quantitative PCR (qPCR) detection after bioinformatics analysis. The miR-204-5p merited further investigation, because it was increased almost 5-fold in diabetic corneal epithelia compared to nondiabetic control corneal epithelia. Using luciferase activity assay, we identified SIRT1 was a direct target of miR-204-5p. The results of flow cytometry and MTT assay demonstrated that downregulation of miR-204-5p increased TKE2 cell growth and restored cell cycle progression in high glucose (HG) conditions by the regulation of Cyclin D1 and p16. Furthermore, we showed downregulation of miR-204-5p promoted HG attenuation of corneal epithelial wound healing via upregulation of SIRT1 in Ins2^Akita/+ mice.

Conclusions.: Our data provide firm evidence of a role for miR-204-5p in the direct regulation of SIRT1 in diabetic corneas and identified the miR-204-5p–mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic keratopathy.

Chinese Abstract

Introduction

Diabetes mellitus (DM) is a chronic disease caused by hyperglycemia, which is attributed to defects in insulin secretion or action.¹ Eye diseases caused by chronic hyperglycemia include diabetic retinopathy (DR), cataract, and keratopathy (corneal pathology). One study reported the presence of corneal epithelial lesions in 47% to 64% of diabetic patients.² Additionally, surgical procedures for DR and/or cataract extraction often are followed by poor healing of the corneal epithelial defects.³ Persistent epithelial defects put patients at greater risk of corneal infection and can lead to corneal ulceration or perforation.⁴ Corneal sensitivity also is decreased in diabetic patients. Diabetic keratopathy is a manifestation of peripheral neuropathy,⁵ and neuronal abnormalities may be the cause of corneal failure in diabetic keratopathy.⁶ Corneal confocal microscopy (CCM) has been used to detect early diabetic peripheral neuropathy (DPN) for the last 10 years.⁷ Currently, few studies have focused on the importance of diabetic keratopathy. 

In DM, gene expression abnormalities are involved in diabetic keratopathy, as molecular abnormalities mediate the loss of the corneal epithelium's ability to regenerate. Control of posttranscription is one of the major mechanisms of gene expression regulation. Our previous study found that the expression of Sirtuin 1 (silent mating type information regulation 2 homolog 1 [SIRT1]) in the corneal tissue of diabetic mice was significantly decreased, and that the overexpression of SIRT1 in the corneal tissue of diabetic mice can significantly promote corneal epithelial wound healing.⁸ The SIRT1 gene is a longevity gene, and is responsible for deacetylating the proteins responsible for overall survival, development, metabolism, and the cell cycle.⁹ It was reported that several posttranscriptional, cell type–specific means control the expression of SIRT1.¹⁰ For example, SIRT1 and forkhead-box class O transcription factors are regulated by transcriptional coactivator p300 in the antioxidant levels of retinal microvascular endothelial cells.¹¹ However, mechanisms that modify the expression of SIRT1 in diabetic corneas are not well understood. 

MicroRNAs (miRNAs or miRs) are a class of small, endogenous, noncoding RNAs that participate in the regulation of gene expression by binding to target mRNA to prevent protein production at the posttranscriptional levels.¹² There is general understanding of the functions of miRNA in multiple biological processes, such as growth, cell death, cell proliferation, central nervous system function, and tumor formation.¹³ At present, several miRNAs, such as miR-195, have been reported to target SIRT1 in diabetic pathological conditions in DR.¹⁴ However, few studies have examined miRNA-mediated regulation of SIRT1 in diabetic keratopathy. 

In the present study, we first used bioinformatics to predict which miRNAs might regulate the expression of SIRT1 (available in the public domain at http://www.targetscan.org/). We then used quantitative real-time PCR (qPCR) technology to validate the expressions of these miRNAs in diabetic corneas. Among these miRNAs, miR-204-5p was identified as being upregulated more than 5.0-fold in diabetic corneas compared to the nondiabetic control. Interestingly, another study in our lab found that the expression of miR-204-5p was downregulated 66.8-fold in human posterior capsule opacification (PCO) tissues compared to those in the normal attached lens epithelial cells (LECs).¹⁵ These data uncovered the multiexpression pattern of miR-204-5p in the eye, suggesting miR-204-5p may be one of the key miRNAs in different diseases of the eye. We then investigated the role of miR-204-5p in regulating SIRT1 in diabetic keratopathy. As SIRT1 has a crucial role in the regulation of the cell cycle and survival,⁹ we investigated whether miR-204-5p–mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic corneas. 

Materials and Methods

Animals

The mice were treated in compliance with the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Use Committee, Shandong Eye Institute, approved the experimental protocol. The C57BL/6J-Ins2^Akita (Ins2^Akita/+) mice and control Ins2^+/+ mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and genotyped according to a protocol.¹⁶ Male mice aged 4 weeks exhibited a progressive loss of β-cell function and significant hyperglycemia,¹⁶ and developed typical chronic complications of diabetes, such as retinopathy, neuropathy, and nephropathy.^17,18 Tail-vein blood glucose concentrations were determined using a commercial glucometer (Ascencia CONTOUR Glucometer; Bayer Diabetes Care, Elkhart, IN, USA). 

Proparacaine hydrochloride (0.5%) was used for topical anesthesia. All animals were anesthetized intraperitoneally with ketamine (37.5 mg/mL) and xylazine (1.9 mg/mL). The in vivo corneal injury model was described previously by Xu et al.¹⁹ The miRNA-specific antagomir was used to downregulate the expression of miR-204-5p (RiboBio Co., Ltd., Guangzhou, China). The mice were divided randomly into three groups, and each group contained 10 mice: group 1 included untreated control Ins2^+/+ mice, group 2 included 2.5 nmol miR-204-5p antagomir–treated Ins2^Akita/+ mice, and group 3 included 2.5 nmol miR-204-5p antagomir native-control (NTC)–treated Ins2^Akita/+ mice. The left eyes of the mice in all groups remained uninjured and untreated for use as normal controls. In group 1, the right eyes were untreated after injury. For groups 2 and 3, miR-204-5p antagomir or antagomir NTC were injected into the subconjunctival site of the right eyes on the same day of corneal epithelium injury. The experiment was performed twice. All groups were assessed using fluorescein sodium staining. Corneal epithelia from the mice were isolated 48 hours after injury and processed for Western blot or qPCR to determine the effects of miR-204-5p antagomir on hyperglycemia-attenuated corneal epithelial wound healing. 

Cell Culture and Treatment

The TKE2 cell line, which is a murine limbal/corneal epithelium-derived progenitor cell line,²⁰ was a kind gift from Wei Li (Xiamen University, Xiamen, China). The TKE2 cells were maintained in keratinocyte serum-free medium (KSFM; Life Technologies, Shanghai, China) supplemented with 2.5 ng/mL human recombinant epidermal growth factor (EGF), 25 μg/mL bovine pituitary extract (BPE), and 1% penicillin in a humidified 5% CO₂ incubator at 37°C. The TKE2 cells were maintained in media containing 5 mM D-glucose (normal glucose [NG]), 25 mM D-glucose (high glucose [HG]), or 5 mmol/L D-glucose plus 20 mmol/L mannitol (high mannitol [HM]). 

We used adenovirus (Ad)-expressing short interfering RNA (siRNA) to knock down the expression of SIRT1 in TKE2 cells. Ad-expressing SIRT1 siRNA and green fluorescent protein (GFP) were purchased from Han Bio-Technology Co., Ltd. (Shanghai, China), and Ad-expressing GFP was used as the negative control. The virus concentration was calculated as 10¹¹ viral particles per ml. 

Transfection

All miRNA mimics, inhibitors, and nontargeting negative controls were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The TKE2 cells were seeded on 6-well plates in completed keratinocyte serum-free (KSFM) medium to 80% confluence. The cells then were shifted to KSFM medium with no EGF and BPE overnight for starvation. Next, the cells were transiently transfected with a miRNA mimic or inhibitor, nontargeting negative control, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 6 hours according to the manufacturer's protocol. The cells then were transferred to KSFM medium and were collected for further analysis 24 hours after transfection. 

qPCR Analysis

To validate the results from bioinformatics analysis, a qPCR analysis of miRNAs was performed using the diabetic or control mice corneal epithelia samples. Total RNA was isolated with a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). All specific primers for miRNA expression were designed and synthesized by RiboBio Co. Ltd. using the mirVana qPCR Primer Sets. The SYBR Green protocol was used on an ABI 7500 system (Applied Biosystems, Foster City, CA, USA) and data analysis was conducted with the SDS system software (7500 System; Applied Biosystems). The levels of an endogenous control, U6 (RiboBio Co.), were used to normalize the expression levels of each miRNA. All reactions were performed in triplicate and included controls without a template for each miRNA. The fold change in miRNA expression was calculated using the comparative CT method. The corneal epithelia samples from nondiabetic control mice were used as a calibrator. The data were presented as the fold change relative to the calibrator. 

For the quantification of SIRT1, cyclin D1, p21, p16, and ribosomal protein L5 (RPL5), cellular RNA was prepared using the TRIzol Reagent (Invitrogen) according to the manufacturer's protocol and was quantified using a spectrophotometer (Eppendorf 1108 spectrophotometer; Eppendorf, Hamburg, Germany). The cDNA was synthesized from 1 μg RNA with oligo (dT)₂₀ primers with an MMLV First-Strand Synthesis Kit (C28025-032; Invitrogen) and measured by qPCR as mentioned above. The primers were designed using commercial software (Primer Express; Applied Biosystems). The primer sequences used in this study are provided in Supplementary Table S1. 

Western Blot Analysis

The samples were homogenized in 100-μL buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 100 μg/mL phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100. The homogenates, which contained 20 μg protein, then were assayed with 15% SDS-polyacrylamide gels (Mini-Protean II system; Bio-Rad Laboratories, Mississauga, ON, Canada) and transferred to polyvinyl difluoride membranes (Thermo Fisher Scientific — China, Beijing, China). The blots were probed with the following primary antibodies: SIRT1 (ab12193; Abcam, Cambridge, MA, USA), Cyclin D1 (A1301; ABclonal, Shanghai, China), p16 (ab51243; Abcam), and p21 (ab7960; Abcam). 

Flow Cytometric Analysis

Cell cycle status in the TKE2 cells was determined by measuring nuclear DNA content. The cells were collected 48 hours after treatment with miR-204-5p antagomir or antagomir NTC, centrifuged at 310g for 5 minutes, and washed twice with 3 mL ice-cold PBS. The cells then were fixed in 70% ethanol at 4°C for more than 4 hours. The pellet was collected by centrifugation before the RNase A solution was added (final concentration 10 mg/mL). After a 1-hour incubation at 37°C, the cells were stained with propidium iodide (PI; final concentration 100 μg/mL) at 37°C for 30 minutes. Flow cytometry was used to analyze the samples with Multi-Cycle software. Three independent experiments were performed in triplicate. 

Analysis of Cell Viability by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyl Tetrazolium Bromide (MTT) Assay

Cell viability was measured by the mitochondrial-dependent reduction of MTT to purple formazan. The cells were incubated with MTT solution (1 mg/mL final concentration) for 4 hours at 37°C. The medium was carefully removed, and formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The extent of the reduction of MTT was determined by measurement of the absorbance at 550 nm. Three independent experiments were performed in triplicate. 

Luciferase Activity Assay

The prediction of the possible miRNAs that could regulate the expression of SIRT1 was performed with the miRNA target prediction algorithms (available in the public domain at http://www.targetscan.org/). Mouse SIRT1 3′-UTR containing the putative target site for miR-204-5p was amplified from genomic DNA by PCR amplification and inserted into the pMIR-REPORT (RiboBio). The mutation from a site of perfect complementarity also was generated by the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer's instructions. The cells were transiently transfected with wild-type or mutant reporter plasmid and miRNA mimic using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured 24 hours after transfection. Three independent experiments were performed in triplicate. 

Statistical Analysis

All results are expressed as the means ± SEM. For the results shown in Figure 1, the difference was calculated using an unpaired t-test. For the other results, statistical analyses were performed using a 1-way ANOVA by comparing the groups using the Student Newman-Keuls test and the least significant difference procedure performed with GraphPad Prism software 5.0 (GraphPad Software, Inc.). A value of P < 0.05 was considered statistically significant. 

Figure 1

 

Validation of selected possible miRNAs which regulate the expression of SIRT1 by qPCR in corneal epithelium of diabetic mice (Ins2^Akita/+) compared to nondiabetic control mice (Ins2^+/+). The expression of mmu-miR-22, mmu-miR-122, mmu-miR-181a, mmu-miR-181b, mmu-miR-200, and mmu-miR-204-5p markedly upregulated in the corneal epithelium of Ins2^Akita/+ mice compared to control Ins2^+/+ mice. Meanwhile, the expression of mmu-miR-138, mmu-miR-181c, and mmu-miR-181d markedly downregulated in the corneal epithelium of Ins2^Akita/+ mice compared to control Ins2^+/+ mice. Values are means ± SEM and expressed relative to internal control (U6, n = 4 for each group; *P < 0.05 versus control). The difference was calculated using an unpaired t-test by GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA).

Results

Identification of miRNA to the Regulation of SIRT1 in Diabetic Corneal Epithelia by qPCR

We used the bioinformatics methods (available in the public domain at http://www.targetscan.org/) to identify which miRNAs might contribute to the regulation of SIRT1 in diabetic keratopathy. We then used qPCR technology to validate the expressions of these miRNAs in diabetic corneal epithelia. The results of the bioinformatics analysis are shown in Supplementary Figure S1. Nine miRNAs were selected for the qPCR detection, as shown in Figure 1. The expression of mmu-miR-22, mmu-miR-122, mmu-miR-181a, mmu-miR-181b, mmu-miR-200, and mmu-miR-204-5p were markedly upregulated in the corneal epithelium of Ins2^Akita/+ mice compared to control Ins2^+/+ mice (n = 4 for each group). Meanwhile, the expression of mmu-miR-138, mmu-miR-181c, and mmu-miR-181d were markedly downregulated in the corneal epithelium of Ins2^Akita/+ mice compared to control Ins2^+/+ mice (n = 4 for each group). One of the upregulated miRNAs, miR-204-5p, merited further investigation because it was increased almost 5-fold in diabetic corneal epithelia compared to nondiabetic control corneal epithelia. A literature search did not reveal any correlation between miR-204-5p and the clinicopathological features of diabetic keratopathy. 

Mir-204-5p Directly Targets and Regulates SIRT1 Protein Levels in Corneal Epithelial Cells

To test the possibility of a direct link between miR-204-5p and SIRT1, TKE2 cells were transfected with the miRNA-204-5p mimic, inhibitor, or NTC. The cells then were collected for qPCR detection or Western blot analysis. After 24 hours of transfection with miRNA mimic, the expression of miR-204-5p was increased 4.16-fold compared to the miRNA mimic NTC (n = 4 for each group), and after transfection with miRNA inhibitor, the expression of miR-204-5p was decreased 1.56-fold compared to the miRNA inhibitor NTC (n = 4 for each group, Fig. 2A). The SIRT1 protein expression was decreased by transfection with the miR-204-5p mimic, but increased by transfection with a miR-204-5p inhibitor in TKE2 cells (n = 3 for each group, Fig. 2B). However, SIRT1 mRNA levels were not significantly influenced by the overexpression or inhibition of miR-204-5p (Fig. 2C), suggesting that SIRT1 expression was primarily inhibited by miR-204-5p at the posttranscriptional level. Furthermore, we performed a dual luciferase reporter assay in the TKE2 cells. The results are shown in Figure 2D. A significant decrease in relative luciferase activity was observed when pMIR-RB-REPORT-SIRT1-3′-UTR was cotransfected with the miR-204-5p mimic (n = 4 for each group). Significantly, the mutation of the perfectly complementary sites in the 3′-UTR of SIRT1 abolished the suppressive effect owing to the disruption of the interaction between miR-204-5p and SIRT1. Together, these results confirmed that SIRT1 is a direct target of miR-204-5p and is regulated by miR-204-5p. 

Figure 2

 

miR-204-5p directly targets and regulates SIRT1 protein levels in corneal epithelial cells. (A) The expression of miR-204-5p in TKE2 cells after transfection with miR-204-5p mimics, inhibitor or NTC. The TKE2 cells with no transfection were used as control (con). Values are means ± SEM (n = 4 for each group, *P < 0.05 versus control). (B) The expression of SIRT1 protein in TKE2 cells after transfection with miR-204-5p mimics, inhibitor or NTC. The TKE2 cells with no transfection were used as control (left: data from the gels; right: normalization to GAPDH). Values are means ± SEM (n = 3 for each group, *P < 0.05 versus control). (C) The expression of SIRT1 mRNA in TKE2 cells after transfection with miR-204-5p mimics, inhibitor or NTC. The SIRT1 mRNA levels were not significantly influenced by the overexpression or inhibition of miR-204-5p, suggesting that SIRT1 expression was primarily inhibited by miR-204-5p at the posttranscriptional level. (D) A dual luciferase reporter assay in TKE2 cells (top: position of the miR-204-5p target sequence in the 3′-UTR region of SIRT1 mRNA; bottom: results of dual luciferase reporter assay). A significant decrease in relative luciferase activity was observed when pmiR-RB-REPORT-SIRT1-3′-UTR was cotransfected with a miR-204-5p mimic. Significantly, the mutation of the perfectly complementary sites in the 3′-UTR of SIRT1 abolished the suppressive effect owing to the disruption of the interaction between miR-204-5p and SIRT1. Values are means ± SEM (n = 4 for each group, *P < 0.05 versus all of other groups).

Effect of Decreased miR-204-5p Expression in Regulation of SIRT1 and Cell Cycle–Related Proteins in TKE2 Cells in HG Conditions

To address whether HG-induced SIRT1 downregulation in corneal epithelia is mediated by miR-204-5p, the TKE2 cells were treated with 5- and 25-mmol/L D-glucose (NG and HG, respectively) for 48 hours. The expression of miR-204-5p was increased following exposure with HG (compared to NG) instead of the HM osmotic control (n = 4 for each group, Fig. 3A). We then transfected TKE2 cells in HG with miR-204-5p inhibitor, and detected the expression of SIRT1, Cyclin D1, p21, and p16. The HG induced the downregulation of SIRT1 and Cyclin D1 and the upregulation of p16 (n = 4 for each group, Fig. 3B). After 48 hours of transfection, the SIRT1 and cyclin D1 mRNA levels were increased by the miR-204-5p inhibitor, while p16 mRNA levels were decreased in TKE2 cells transfected with the miR-204-5p inhibitor in HG conditions (n = 4 for each group, Fig. 3B). Similarly, SIRT1 and cyclin D1 protein expression increased when the TKE2 cells were transfected with the miR-204-5p inhibitor, and the protein expression of p16 decreased when the TKE2 cells were transfected with the miR-204-5p inhibitor in HG conditions (n = 4 for each group, Fig. 3C). However, the expression of p21 (message/protein) showed no significant difference among TKE2 cells transfected with miR-204-5p inhibitor and the other groups (NG, HG, and miR-204-5p inhibitor NTC treatment). These results clearly showed the effect of decreased miR-204-5p expression in the regulation of SIRT1 and cell cycle–related proteins in TKE2 cells in HG conditions. 

Figure 3

 

High glucose induced SIRT1 downregulation in TKE2 cells is mediated by miR-204-5p. (A) The expression of miR-204-5p was increased following exposure with HG (compared to NG) instead of HM (osmotic control) in TKE2 cells. The NG was used as control. Values are means ± SEM (n = 4 for each group, *P < 0.05 versus control). (B) The mRNA expression of SIRT1, cyclin D1, p21, and p16 after TKE2 cells were transfected with miR-204-5p inhibitor. The NG was used as control. Values are means ± SEM (n = 4 for each group, *P < 0.05 versus NG and &P < 0.05 versus HG). (C) Protein expression of SIRT1, cyclin D1, p21, and p16 after TKE2 cells were transfected with miR-204-5p inhibitor. Left: The representative gel results. Right: The graphs used for normalization to GAPDH. The NG was used as control. Values are means ± SEM (n = 4 for each group, *P < 0.05 versus NG and &P < 0.05 versus HG).

Downregulation of miR-204-5p Increases TKE2 Cell Growth, Restores Cell Cycle Progression in HG Conditions, and Changes the Senescence Associated β-Galactosidase (SA-β-Gal) Staining

Since downregulation of miR-204-5p increases the expression of SIRT1 and Cyclin D1, we proceeded to investigate whether downregulation of miR-204-5p affects cell growth. The cells were treated with miR-204-5p antagomir, and the MTT assay was used to determine relative cell growth as described previously.²¹ Data from the MTT colorimetric assay are shown in Figure 4. The absorbances were 0.940 ± 0.028, 0.841 ± 0.067, 0.258 ± 0.051, 0.412 ± 0.032, and 0.845 ± 0.008 in the NG, HM, HG, HG plus miR-204-5p antagomir NTC, and HG plus miR-204-5p antagomir groups, respectively (n = 3 for each group). There were fewer viable cells in the HG group than in the other groups. The absorbances of the miR-204-5p antagomir group were significantly different from those of the HG groups (P < 0.0001), but were not significantly different from those of the NG group (P > 0.05). These data indicated that TKE2 cell growth increased most significantly in the treatment with miR-204-5p antagomir in HG conditions. 

Figure 4

 

Downregulation of miR-204-5p increases TKE2 cell growth by MTT assay. The TKE2 cells were treated with miR-204-5p antagomir and the MTT assay was used to determine relative cell growth. The absorbances were 0.940 ± 0.028, 0.841 ± 0.067, 0.258 ± 0.051, 0.412 ± 0.032, and 0.845 ± 0.008 in the NG, HM, HG, HG+miR-204(A) NTC, and HG+miR-204(A) groups, respectively (n = 3 for each group). There were fewer viable cells in the HG group than other groups. The absorbances of the miR-204-5p antagomir group were significantly different from HG groups (P < 0.0001), but were not significantly different from the NG group (ns, P > 0.05).

Treatment of TKE2 cells with HG resulted in greater inhibition of cell growth than the NG or HM treatment. This may be due to an arrest of cells at specific check points in the cell cycle. Next, we studied the cell cycle status in TKE2 cells by flow cytometric analysis after miR-204-5p antagomir treatment. The S-phase arrest occurred after treatment with HG manifested as the cell percentage in the S phase (19.44 ± 2.50%, n = 4 for each group). Cell cycle analysis showed that the S-phase cell population significantly increased (29.36 ± 2.67%) after miR-204-5p antagomir treatment, compared to other groups (30.20 ± 0.24%, 28.32 ± 0.25%, 19.44 ± 2.50%, and 21.72 ± 0.12% in the NG, HM, HG, and HG plus miR-204-5p antagomir NTC groups, respectively; n = 4 for each group, Fig. 5). 

Figure 5

 

Inhibition of miR-204 rescues the HG induced S phase cell cycle arrest in TKE2 cells. (A) The TKE2 cells were treated with miR-204-5p antagomir and then stained with propidium iodide. The DNA content was analyzed by flow cytometry. The cell cycle phase is indicated by G1, S, and G2. (B) The S phase arrest occurred after treatment with HG manifested as the cell percentage in S phase (19.44 ± 2.50%). Cell cycle analysis showed that the S phase population was significantly increased in cell percentage (29.36 ± 2.67%) after miR-204-5p antagomir treatment, compared to other groups (30.20 ± 0.24%, 28.32 ± 0.25%, 19.44 ± 2.50%, and 21.72 ± 0.12% in the NG, HM, HG, and HG plus miR-204-5p antagomir NTC groups, respectively). Values are means ± SEM (n = 4 for each group, *P < 0.05 versus NG and &P < 0.05 versus HG). Value miR-204-5p (A) means miR-204-5p antagomir.

Because SIRT1 is a longevity gene, we then checked the change of senescence marker expression, SA-β-Gal staining, since HG-induced differentiation (senescence). The representative results of SA-β-Gal staining were shown in Figure 6A. Compared to the NG or HM group, SA-β-Gal–positive cells were observed in the HG group. However, when the cells were treated with miR-204-5p antagomir, a few SA-β-Gal–positive cells were observed in HG conditions, compared to the HG+miR-204(A) NTC or HG group (n = 4 for each group, Fig. 6B). 

Figure 6

 

The change of senescence-associated SA-β-Gal staining in TKE2 cells after downregulation of miR-204-5p in HG conditions. (A) Representative results of SA-β-Gal staining. Compared to the NG group or HM (osmotic control), SA-β-Gal–positive cells were observed in the HG group (arrows). However, when the cells were treated with HG+miR-204(A), a few SA-β-Gal–positive cells were observed in HG conditions, compared to the HG+miR-204 (A) NTC or HG groups (arrows). Scale bar: 50 μm. (B) The SA-β-Gal staining from each group, shown as the number of positive cells counted in a total 10 visual fields for each sample. The numbers of SA-β-Gal–positive cells were decreased in the miR-204-5p antagomir–treated group (also in HG conditions), compared to the HG+miR-204 (A) NTC or HG group (n = 4 for each group).

Downregulation of miR-204-5p Promotes HG Attenuation of Corneal Epithelial Wound Healing Via SIRT1 Regulation In Vivo

To investigate whether downregulation of miR-204-5p affects the attenuation of corneal epithelial wound healing, we examined the effects of miR-204-5p antagomir on corneal epithelial debridement wound healing using type 1 diabetic Ins2^Akita/+ mice and control Ins2^+/+ mice. The miR-204-5p antagomir was injected into the subconjunctival site on the same day of corneal epithelium injury, and the corneal surface wound was monitored using fluorescein dye. The corneas were harvested 48 hours after injection. Based on measurements of the fluorescein-stained areas at 24 and 48 hours after injury, we found that downregulation of miR-204-5p in corneal epithelia promoted the wound healing process (Fig. 7A). At 48 hours after injury, the wound areas in mice corneas administered miR-204-5p antagomir were significantly reduced relative to the wound areas in the antagomir NTC-treated corneas (Fig. 7B). The qPCR analysis revealed that the level of SIRT1 and Cyclin D1 expressions were significantly higher, and the levels of p16 were significantly lower after antagomir treatment (n = 4 for each group, Fig. 8A). Furthermore, Western blotting analysis revealed that the levels of SIRT1 and Cyclin D1 expressions were significantly higher, and the levels of p16 were significantly lower after antagomir treatment (n = 4 for each group, Fig. 8B). Thus, we speculated that the decrease of miR-204-5p may improve corneal epithelial wound healing in diabetic mice via SIRT1 regulation. 

Figure 7

 

The effects of miR-204-5p antagomir on corneal epithelial debridement wound healing in Ins2^Akita/+ mice. (A) The fluorescein-stained corneas at 24, 48, and 72 hours after injury. (B) The remaining wound area at 24, 48, and 72 hours after injury. At 48 hours after injury, the wound area in mice that were administered miR-204-5p antagomir was significantly reduced relative to the wound areas in the antagomir NTC–treated. The image shows the remaining wound area at 24, 48, and 72 hours after injury. Changes in the mean remaining wound areas were calculated using commercial editing software (n = 4 for each group, *P < 0.05).

Figure 8

 

The effect of regulation of the SIRT1, Cyclin D1, p21, and p16 on corneal wound healing in mice after miR-204-5p inhibition. (A) The results of qPCR. (B) The results of Western blot. Left: The representative gel results. Right: The graphs used for normalization. The levels of SIRT1 and Cyclin D1 (message/protein) were significantly higher and the levels of p16 (message/protein) were significantly lower after antagomir treatment. Values are means ± SEM (n = 4 for each group, *P < 0.05 and ns, P > 0.05).

Discussion

Using computer algorithms, we predicted a set of miRNA that regulate SIRT1, and we identified potential miRNA modulators of SIRT1 during diabetic keratopathy. Of the nine regulated miRNAs, miR-204-5p piqued our interest not only because it was significantly upregulated, but also because of its multiple roles in the eyes, such as that of human PCO.¹⁵ Few published studies have focused on characterizing miR-204-5p expression in the normal cornea. Ryan et al.²² compared the miRNA expression profile of normal mouse corneal epithelium to mouse footpad epithelium using microarrays. They found that miR-204 were high in the cornea and low in the footpad. Several reports have demonstrated the possibility of a widespread functional role of miR-204. It is highly expressed in fetal RPE, lens epithelium, and nonpigmented epithelium of the ciliary body.¹⁶ The role of miR-204 in corneal epithelium remains elusive. In this study, we found that miR-204 was highly expressed in diabetic corneal epithelium, and that downregulation of miR-204 promotes HG attenuation of corneal epithelial wound healing via SIRT1 regulation. 

One of the most important roles of miR-204 is that of inhibitor of epithelial-to-mesenchymal transition (EMT) by targeting several key factors in the TGF-β signaling pathway, such as prostaglandin-endoperoxide synthase 2,¹⁶ SMAD4,¹⁶ TGF-β receptor 2, and Snail2.²³ Recent studies by Zhang et al.²⁴ found that miR-204 downregulates SIRT1 and reverts SIRT1-induced EMT in gastric cancer cells. During the process of EMT, they found that the expression of miR-204 was decreased, and upregulation of miR-204 reverted EMT by targeting SIRT1. These results are partly in agreement with the results from our study; that is, the target gene of miR-204 is, indeed, SIRT1, whereas in the pathology of diabetic keratopathy, we found that the expression of miR-204 was upregulated. These patterns of expression of miR-204-5p demonstrated the multiple roles of miR-204 in different conditions, suggesting that miR-204-5p and its target gene, SIRT1, may have important roles in the pathogenesis of diabetic keratopathy. 

There have been several recent reports on the miRNA regulation of SIRT1. The first of these showed that the miRNA miR-34a suppresses SIRT1 in the regulation of apoptosis.²⁵ It also revealed that miR-217 is a miRNA modulator of SIRT1 in the cardiovascular system, that overexpression of miR-217 suppresses SIRT1 expression in young endothelial cells, and that inhibition of miR-217 promotes cellular senescence in older endothelial cells.¹¹ In glucose metabolism, two miRNAs, miR-9 and miR-132, were shown to control SIRT1 in pancreas²⁶ and adipose tissues.²⁷ Ramachandran et al.²⁶ found that glucose-stimulated insulin secretion is accompanied by changes in miR-9, which is mediated by SIRT1. Strum et al.²⁷ found that, in low-serum conditions, miR-132 expression is upregulated, and overexpression of miR-132 blocks deacetylation of p65/NFκB, causing induction of IL-8 and monocyte chemoattractant protein-1 (MCP-1) by targeting SIRT1. 

Jaliffa et al.²⁸ examined the localization of SIRT1 in adult mouse eyes and they found SIRT1 is localized in the nucleus and cytoplasm of corneal epithelial cells, and in the nucleus of keratocytes and corneal endothelial cells. Alves et al.²⁹ reported that, in the normal human corneal epithelium, 50% showed negative expression of SIRT1 and 30% weak expression, and 20% were considered significantly immunoreactive using 10 corneal specimens. Among diabetic patients, wound healing is slow. In diabetic keratopathy, once the cornea has been damaged, delayed healing of the epithelial wounds can be very difficult to manage clinically and often is followed by greater risk of long-lasting epithelial erosion with poor healing of the epithelial defects.⁶ The regulation of cell migration and proliferation have a vital role in corneal epithelial wound healing, and basal cells outside the wound area are stimulated by the epithelial debridement to enter the cell cycle.³⁰ In this study, we found that miR-204 negatively regulated SIRT1 expression and downregulation of miR-204 promoted epithelial wound healing in diabetic cornea via the S transition cell cycle. 

In normal glucose conditions, the expression of SIRT1 mRNA was not changed, when the TKE2 cells treated with miR-204-5p agomir or antagomir (Fig. 2C). However, in HG conditions, when the TKE2 cells were transfected with miR-204-5p inhibitor, the expression of SIRT1 mRNA was upregulated (Fig. 3B). Additionally, we found that when the diabetic mice were treated with miR-204-5p antagomir through subconjunctival injection, the expression of SIRT1 mRNA also was upregulated (Fig. 8A), suggesting the specific role of miR-204-5p in HG conditions. 

After the TKE2 cells were treated with HG, the cell cycle was arrested in the S phase, indicating that HG induced the cell cycle disorders in TKE2 cells and decreased the ability of cell division. Meanwhile, the expression of cyclin D1 and p16 was downregulated. This finding is partly consistent with other recent reports. Lee et al.³¹ found that loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycemia. When the expression of miR-204 was inhibited by antagomir in TKE2 cells, the cell cycle arrest induced by HG was rescued. We also found that the expression of cyclin D1 was upregulated and the expression of p16 was downregulated. Factor p16 is one of the most important cell cycle–related factors, and overexpression of p16 can cause premature aging and loss of function in stem cells.³² In addition, the expressions of p21 and cycle E1 (data not shown) showed no significant difference in TKE2 cells between miR-204 antagomir treatment and antagomir NTC treatment in HG conditions. These results suggested that the cyclin D1-CDK4 may be one of the major pathways in the TKE2 cell cycle regulation of miR-204 in HG conditions by targeting SIRT1. 

In summary, this study described a role for the miR-204 in the regulation of SIRT1 in diabetic corneal epithelial wound healing. Using a combination of in vitro and in vivo approaches, we demonstrated that hyperglycemia increased miR-204 expression and induced cell cycle arrest in the S phase in corneal epithelial cells. In addition, we showed that miR-204 inhibition could overcome the detrimental corneal wound healing effects of hyperglycemia in association with an increase of SIRT1 and restoration of the cell cycle. Although inhibition of miR-204 might rescue diabetic mice after corneal injury, many downstream target genes of miR-204 may be responsible for modified wound healing of patients with diabetic keratopathy. Further studies should focus on the activation of other downstream targets and pathways of miR-204 in diabetic keratopathy. 

Acknowledgments

Supported by the State Key Basic Research (973) Project of China Grant 2012CB722409, the National Natural Science Foundation of China Grants 81370990 and 30901637, and the Shandong Province Natural Science Foundation Grant BS2012YY030. The authors alone are responsible for the content and writing of the paper. 

Disclosure: J. Gao, None; Y. Wang, None; X. Zhao, None; P. Chen, None; L. Xie, None 

Footnotes

 JG and YW contributed equally to the work presented here and should therefore be regarded as equivalent authors.