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Glaucoma  |   April 2014
TGF-β1 Stimulates Human Tenon's Capsule Fibroblast Proliferation by miR-200b and its Targeting of p27/kip1 and RND3
Author Affiliations & Notes
  • Jun Tong
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Yuxuan Fu
    Department of Physiology, Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Xinyu Xu
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Shuxin Fan
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Hong Sun
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Ya Liang
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Kai Xu
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Zhilan Yuan
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Yingbin Ge
    Department of Physiology, Nanjing Medical University, Nanjing, Jiangsu Province, China
  • Correspondence: Zhilan Yuan, Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing, 210029, Jiangsu Province, China; zhilanyuan@vip.sina.com.  
  • Yingbin Ge, Department of Physiology, Nanjing Medical University, Hangzhong Road 140, Nanjing, Jiangsu Province, China; ybge@njmu.edu.cn
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2747-2756. doi:10.1167/iovs.13-13422
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      Jun Tong, Yuxuan Fu, Xinyu Xu, Shuxin Fan, Hong Sun, Ya Liang, Kai Xu, Zhilan Yuan, Yingbin Ge; TGF-β1 Stimulates Human Tenon's Capsule Fibroblast Proliferation by miR-200b and its Targeting of p27/kip1 and RND3. Invest. Ophthalmol. Vis. Sci. 2014;55(4):2747-2756. doi: 10.1167/iovs.13-13422.

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

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Abstract

Purpose.: To evaluate the role of miR-200b expression in the proliferation of human Tenon's capsule fibroblasts (HTFs) induced by transforming growth factor-beta 1 (TGF-β1).

Methods.: Human Tenon's capsule fibroblasts were treated with various doses of TGF-β1 for 24 hours. Cell proliferation was quantified by the cell counting kit-8 (cck-8) assay, cell cycle analysis, 5-ethynyl-2-deoxyuridine (EdU) assay, and analysis of cyclin E, cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. MicroRNA-200b (miR-200b) was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by the luciferase assay and Western blot analysis. The effect of miR-200b on the proliferation of HTFs was analyzed using both miR200b-mimic and inhibitor-transfected HTFs and confirmed in p27/kip1 and RND3 (the target of miR-200b) knockdown cells.

Results.: The proliferation of the TGF-β1–treated HTFs increased significantly in a dose- and time-dependent manner. Treatment with 5 ng/mL TGF-β1 caused an upregulation of miR-200b. The luciferase assay identified p27/kip1 and RND3 as target genes for miRNA-200b, which was confirmed by the expression of p27/kip1 and RND3 and their downstream products (cyclinE and cyclinD1) in the TGF-β1–treated cells. Transforming growth factor-β1 and miR-200b mimics enhanced the proliferation of HTFs; suppressed the expression of p27/kip1 and RND3; and subsequently stimulated the expression of cyclinE, cyclinD1, and PCNA. The miR-200b inhibitor attenuated the effects of TGF-β1 on HTFs. Furthermore, knockdown of p27/kip1 and RND3 resulted in an increase in cell proliferation and expression of the proliferation-related genes.

Conclusions.: MicroRNA-200b acts as a stimulant for the proliferation of HTFs by targeting p27/kip1 and RND3.

Introduction
Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by the development of a specific type of optic neuropathy and visual field loss. Reduction of intraocular pressure (IOP) is the mainstay of treatment to arrest the progression of this potentially blinding disease. 1,2 A channel for the drainage of aqueous humor from the anterior chamber to the subconjunctival space is created during glaucoma filtration surgery (trabeculectomy). This channel is essential for controlling the IOP, which in turn appears to be effective in maintaining the patency of the aqueous drainage channel. 35 One of the main reasons for the failure of trabeculectomy is excessive subconjunctival fibrosis (scar formation) due to the proliferation of Tenon's fibroblasts and the subsequent increase in the synthesis of extracellular matrix (ECM) and collagen contraction. 6,7 Although transforming growth factor-beta 1 (TGF-β1) is known to play a crucial role in scar formation, the detailed mechanisms associated with its function have not yet been elucidated. 8  
Several recently published papers have demonstrated the regulatory effects of microRNA (miRNA) on TGF-β and have rekindled interest in TGF-β–mediated fibrosis. 911 MicroRNAs are single-stranded, non-coding RNA molecules, approximately 22 nucleotides in length, which function as repressors of gene expression through mRNA cleavage and cause translational repression by base-pairing with the 3′-untranslated regions (UTRs) of miRNAs, thereby inhibiting translation or promoting degeneration of miRNAs. MicroRNAs are involved in the regulation of cell proliferation, apoptosis, differentiation, and metabolism. 12,13 For instance, miR-24 downregulates the TGF-β1 activity in human trabecular meshwork cells, and miRNA-29b suppresses the type I collagen gene by repressing the PI3K/Akt/Sp1 pathway in human Tenon's capsule fibroblasts (HTFs). 14,15 In contrast, various miRNAs have been shown to be regulated by TGF-β1 and are involved in the proliferation, migration, and apoptosis of cells such as HTFs. 16,17  
According to the microarray data presented by Li et al., 15 miR-200b was upregulated in the TGF-β1–treated HTFs. Using three different algorithms, we predicted that miR-200b has a binding site on CDKN1B (p27/kip1) and RND3 (RhoE) 3′-UTRs. CDKN1B (p27/kip1) is a negative regulator of the cell cycle and is tightly associated with cyclin E; cyclin D1, which is downstream in the RND3 (RhoE) cascade. 1821 Both cyclin E and cyclin D1 are involved in cell proliferation. It has been reported that miR-200b may be involved in tumor cell proliferation in several types of cancer, such as breast, 22 liver, 20 and ovarian 23 cancers. In this study, we examined the effect of miR-200b on the proliferation of HTFs. We demonstrated that miR-200b induces proliferation of HTFs, in part by targeting CDKN1B (p27/kip1) and RND3 (RhoE). 
Materials and Methods
Cell Culture and Treatment
HTFs, which were established by Wei Tang as previously described in accordance with the tenets of the Declaration of Helsinki. 24 In brief, the Tenon's capsule tissue was cut into small pieces, and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 0.1 U/L penicillin, and 50 μg/mL streptomycin (Gibco, Carlsbad, CA, USA) at 37°C in 5% CO2. After 4 to 7 days, cells of spindle-shaped, stellate and other irregular forms showed surrounding the adherent human Tenon's tissue. After a confluent monolayer of the primary culture stage was obtained, the tissues were discarded, the cells were incubated with 0.05% trypsin and 5 mM EDTA at 37°C for 3 minutes, and transferred to another culture dish. Cells of passage 3 through 6 were used in the subsequent studies. After approximately 80% confluence, the cells were starved for 24 hours and treated with TGF-β1 (PeproTech, London, UK) at various doses and for various durations. 
Immunofluorescence
Cells of passage 3 were seeded in six-well plates and incubated overnight at 37°C to allow the cells to adhere; cells would confluence approximately 80% and then cells were fixed in 4% paraformaldehyde. Nonspecific protein binding sites were blocked with 3% bovine serum albumin (Sigma-Aldrich, Tokyo, Japan). Sections were permeabilized in 0.1% Triton X-100 and probed with rabbit–anti-vimentin (1:400; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit-anti-keratin (1:200; Cell Signaling Technology, Inc., Danvers, MA, USA) or mouse-anti-fibroblast surface protein (FSP, 1:200; Abcam, Cambridge, MA, USA) for overnight at 4°C. The primary antibody was detected with goat–anti-mouse-Alexa 594, goat–anti-rabbit–Alexa 594 conjugated antibodies (1:1000; Invitrogen) or goat–anti-mouse–Alexa 568 for 1 hour at room temperature. Nuclei were detected by co-stain with 4′-6-diamidino-2-phenylindole (DAPI; Invitrogen). At least 50 cells were randomly selected from a single captured field. Cells were observed and photographed via fluorescence microscopy (BX51; Olympus Corporation of the Americas, Tokyo, Japan). Images were analyzed by commercial software (Olympus Cell software; Olympus Corporation of the Americas). 
Cell Counting Kit-8 (CCK-8) Assay
Cell proliferation was measured using the CCK-8 assay kit (Dojindo Laboratories, Kumamoto, Japan). Cells were seeded in 96-well plates in triplicate, and various treatments were applied to the wells. After these treatments, the CCK-8 solution (10 μL) was added to each well, and the wells were incubated for another 2 hours at 37°C. Absorbance was measured at 450 nm (ELx800; BioTek, Winooski, VT, USA). 
5-ethynyl-2-deoxyuridine (EdU) Incorporation Assay
Cell proliferation was also measured using the imaging kits (Click-iT EdU; Invitrogen) according to the manufacturer's instructions. Briefly, cells were seeded in 24-well plates in triplicate, and various treatments were applied. After these treatments, cells were labeled for 8 hours with 10 μmol/L EdU at 37°C. After fixing the cells with 3.7% formaldehyde and permeabilizing them with 0.5% Triton X-100 in PBS, the cells were incubated for 30 minutes with azide-conjugated AlexaFluor 488 dye in TBS supplemented with 4 mM CuSO4 and in PBS for 20 minutes at room temperature. The cell photographs were captured by a fluorescent microscope (Olympus Corporation of the Americas) and Java-based imaging software (ImageJ; National Institutes of Health, Bethesda, MD, USA). At least 50 cells were randomly selected from a single captured field, and the intensity of the average nuclear fluorescence was calculated. This assay was performed in three independent experiments. 
Cell Cycle Analysis
After various treatments, cells were harvested and washed with ice-cold PBS and fixed with 70% ethanol overnight. Fixed cells were rehydrated in PBS for 10 minutes and subjected to PI/RNase staining followed by flow cytometry analysis using a cytometer (BD FACSCalibur; BD Biosciences, San Jose, CA, USA) using commercial software (Cell Quest Pro; BD Biosciences). 
Western Blotting
Treated cells were rinsed three times with ice-cold PBS and lysed by RIPA buffer (Beyotime, Hangzhou, China) according to the manufacturer's instructions. The lysates were centrifuged at 12,000g at 4°C for 10 minutes, and the supernatants were submitted to Western blotting. The protein concentration was measured using a protein assay kit (BCA Protein Assay Kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts (25 μg/lane) of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5% skim milk in TBST at room temperature for 2 hours and then incubated with primary antibodies at 4°C overnight. The membranes were subsequently incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG (diluted 1:5000; Santa Cruz Biotechnology, Inc.) for 1 hour. Membranes were exposed using the ECL system (Millipore Corp.). The primary antibodies used were mouse monoclonal anti-p27 (BD Biosciences); mouse monoclonal anti-RhoE (R&D Systems, Minneapolis, MN, USA); mouse monoclonal anti-cyclin D1 and cyclinE (Santa Cruz Biotechnology, Inc.); and rabbit polyclonal anti-proliferating cell nuclear antigen (PCNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, Inc.). Expression levels were normalized to GAPDH. 
RNA Preparation and Quantitative Real-Time PCR
Total RNA and miRNA in treated cells were isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The stem-loop reverse-transcription (RT) primers were designed as follows: miR-200b, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCATCAT-3′, and U6, 5′-CGCTTCACGAATTTGCGTGTCA-3.′ Reverse transcription was performed using a DNA synthesis kit (RevertAidTM First Strand cDNA Synthesis Kit; Fermentas, Ottawa, Canada) following the manufacturer's instructions. The random RT primer 5′- (dN) 9-3′ was used for the p27/kip1, RND3, and GAPDH genes. The polymerase chain reaction (PCR) primers were designed as follows: miR-200b sense, 5′-GCGGCTAATACTGCCTGGTAA-3′, reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 sense, 5′-CGCTTCGGCAGCACATATACTA-3′, reverse, 5′-CGCTTCACGAATTTGCGTGTCA-3′; P27 sense, 5′-TAATTGGGGCTCCGGCTAACT-3′, reverse, 5′-TTGCAGGTCGCTTCCTTATTC-3′; RND3 sense, 5′-ATAGAGTTGAGCCTGTGGGACAC-3′, reverse, 5′-AGGGTCTCTGGTCTACTGATGTC-3′; GAPDH sense, 5′-TGCACCACCAACTGCTTAGC-3′, reverse, 5′-GGCATGGACTGTGGTCATGAG-3′ using an master mix (SABI SYBR Green Master Mix; Invitrogen) according to the manufacturer's protocol. All mRNA quantification data were normalized to human GAPDH, and U6 snRNA was used as an endogenous control for the miRNA detection. The data were processed using 2-ΔΔCt methods. 
Cell Transfections and Infection
All of the human miRNA mimics, inhibitors, and their scramble controls were designed by and purchased from RiBo Biological Co., Ltd. (Guangzhou, China). The miRNAs were transfected into HTFs using a commercial (Lipofectamine2000; Invitrogen) according to the manufacturer's guidelines. The final concentrations of miRNA mimics and miRNA inhibitors were 100 and 200 nM, respectively. To establish stable knockdown cell lines, the lentivirus vectors pLV-p27/kip1-inhibitor (target sequences: 5′-AAGGTTGCATACTGAGCCAAG-3′) and pLV-RhoE-inhibitor (target sequences: 5′-TAAGTAGAGCTCTCCAATCA-3′), along with the packaging plasmid mix, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Individual plasmids and the packaging mix were cotransfected into HEK293TN packaging cell lines using a commercial reagent (Invitrogen). The culture supernatant was concentrated using a lentiviral solution (Lenti-Pac Lentivirus Concentration Solution; GeneCopoeia, Inc., Guangzhou, China). The control plasmid provided with the lentivirus kit was processed as described above to obtain the control pseudovirus (sequence: 5′-GTCCCGGATACCTAATAAA-3′). HTFs were incubated with the lentiviruses in the presence of 2 μg/mL polybrene (Gibco) and were cultured with 2 μg/mL puromycin (Sigma-Aldrich) for at least 96 hours to select the stably transfected cells. 
miRNA Target Prediction
Internet databases, such as microRNA.org (www.microrna.org, in the public domain), TargetScan (www.targetscan.org, in the public domain), and PITA (http://genie.weizmann.ac.il/pubs/mir07, in the public domain), were used to predict downstream targets of the significantly changed miRNAs. Each target was regarded as positive only if it was predicted by at least two of the three algorithms. 
Generation of p27/kip1 and RND3 3′-UTR Reporter Constructs and Luciferase Activity Assays
The p27/kip1 and RND3 3′-UTR reporter constructs were generated from Shenzhen Pingkang Biological Co., Ltd. (Shenzhen, China). Briefly, the cDNA fragment of the human p27/kip1 and RND3 mRNA 3′-UTR, which contains the predicted miR-200b target site, was obtained by RT-PCR and cloned into the pLUC-luciferase (pLUC-Report plasmids; Ambion, Inc., Austin, TX, USA), downstream of the luciferase coding sequence. One mutant construct containing seven nucleotide mutations at the predicted miR-200b binding site region (1022–1027) in human p27/kip1 mRNA 3′-UTR and two mutant constructs containing seven nucleotide mutations at the predicted miR-200b binding site region (1584–1591 or 1729–1735) in human RND3 mRNA 3′-UTR were created by the PCR mutagenesis method. Human Tenon's capsule fibroblasts were seeded in 24-well plates at a density of 5 × 104 cells/well and cotransfected with the control or miR-200b mimic, 0.2 μg of pLUC-luciferase (Ambion, Inc.), and 0.04 μg of pRL-TK internal control (Promega, Madison, WI, USA) plasmids. Twenty-four hours after transfection, luciferase activities were measured using an assay system (Dual-Luciferase Reporter Assay System; Promega) according to the manufacturer's instructions. 
Statistical Analysis
Data from at least three independent experiments are presented as the means ± standard error of the mean (SEM) using statistical software (SPSS 19.0; SPSS, Inc., Chicago, IL, USA). The differences between treatment groups were analyzed using Student's t-test or ANOVA, followed by the Student-Newman-Keuls (SNK) test. P values from a two-tailed test <0.05 were considered statistically significant. 
Results
Fibroblasts in Identification
Cells of spindle-shaped, stellate, and other irregular forms showed surrounding the adherent human Tenon's tissue after 4 to 7 days. After passage, cells migrated to the left space and would confluence approximately 90% or more (Fig. 1A). 
Figure 1
 
Fibroblasts in identification. Cells migrated from the adherent human Tenon's tissue after 4 to 7 days and cells confluenced 90% or more after passages. (A) Pictures were taken at 100× magnification. Human Tenon's fibroblasts cells were incubated with vimentin, cytokeratin, and fibroblast surface protein antibodies. (B) Immunofluorescence with vimentin-positive staining (green), and nuclear staining with DAPI (blue). (C) Immunofluorescence with FSP-positive staining (red), and nuclear staining with DAPI (blue). (D) Negative cytokeratin staining (green), and nuclei labeled with DAPI (blue). All pictures were taken at 400× magnification.
Figure 1
 
Fibroblasts in identification. Cells migrated from the adherent human Tenon's tissue after 4 to 7 days and cells confluenced 90% or more after passages. (A) Pictures were taken at 100× magnification. Human Tenon's fibroblasts cells were incubated with vimentin, cytokeratin, and fibroblast surface protein antibodies. (B) Immunofluorescence with vimentin-positive staining (green), and nuclear staining with DAPI (blue). (C) Immunofluorescence with FSP-positive staining (red), and nuclear staining with DAPI (blue). (D) Negative cytokeratin staining (green), and nuclei labeled with DAPI (blue). All pictures were taken at 400× magnification.
Immunofluorescence staining was used to identify fibroblasts. Vimentin antibody staining showed cytoplasm as green fluorescence, with a large majority of fibroblasts blue-stained nuclei (Fig. 1B). Fibroblast surface protein (FSP) antibody staining showed cytoplasm as red fluorescence, with fibroblasts blue-stained nuclei (Fig. 1C). Cytokeratin antibody staining was negative, with cells blue-stained nuclei (Fig. 1D). 
TGF-β1 Promotes Proliferation of HTFs
As previously reported, TGF-β1 was found to be a potent promoter of fibroblast growth. 25,26 To clarify the effect of TGF-β1 on HTFs, these cells were treated with TGF-β1 at concentrations of 0, 0.5, 1.0, 2.0, 5.0, and 10 ng/mL for 24 hours, or for 0, 12, 24, and 48 hours at a TGF-β1 dose of 5 ng/mL. The cell proliferation increased in a dose-dependent manner, as evidenced by the results obtained from the CCK-8 assay; and the median effective dose (ED50) of TGF-β1 was 5 ng/mL (Fig. 2A). These results were confirmed by the cell cycle analysis, which demonstrated that the number of cells in S phase also increased in a dose-dependent manner (Fig. 2B). In the HTFs treated with 5 ng/mL of TGF-β1, the results of the CCK-8 assay revealed a time-dependent cell proliferation (Fig. 2C), which was confirmed by the expression of the markers of cell proliferation—that is, cyclin D1, cyclin E, and PCNA (Fig. 2D). All these results indicate that TGF-β1 stimulated the proliferation of the HTFs in a dose- and time-dependent manner. 
Figure 2
 
Transforming growth factor-β1 promotes HTF proliferation. Human Tenon's capsule fibroblasts were treated with TGF-β1 for 24 hours at doses of 0, 0.5, 1.0, 2.0, 5.0, and 10 ng/mL, and cell proliferation was detected using the CCK-8 assay (A) and cell cycle analysis (B). Human Tenon's capsule fibroblasts were treated with TGF-β1 at a dose of 5.0 ng/mL for 0, 12, 24, and 48 hours, and cell proliferation was detected using the CCK-8 assay (C) and by assessing the expression of cell proliferation markers (cyclin D1, cyclin E, and PCNA [D]). *P < 0.05 versus controls. **P < 0.01 versus controls.
Figure 2
 
Transforming growth factor-β1 promotes HTF proliferation. Human Tenon's capsule fibroblasts were treated with TGF-β1 for 24 hours at doses of 0, 0.5, 1.0, 2.0, 5.0, and 10 ng/mL, and cell proliferation was detected using the CCK-8 assay (A) and cell cycle analysis (B). Human Tenon's capsule fibroblasts were treated with TGF-β1 at a dose of 5.0 ng/mL for 0, 12, 24, and 48 hours, and cell proliferation was detected using the CCK-8 assay (C) and by assessing the expression of cell proliferation markers (cyclin D1, cyclin E, and PCNA [D]). *P < 0.05 versus controls. **P < 0.01 versus controls.
Effect of TGF-β1 on miR-200b Expression by the HTFs
To identify the effects of TGF-β1 on the expression of miR-200b by the HTFs, we treated the HTFs with 5 ng/mL of TGF-β1 for 24 hours and then applied qRT-PCR to detect the levels of miR-200b. As described in Figure 3, the level of miR-200b in the TGF-β1–treated HTFs was 2-fold higher than that in the control HTFs. 
Figure 3
 
Transforming growth factor-β1 enhances miR-200b expression. MicroRNA-200b levels were assessed by qRT-PCR and determined using the 2-ΔΔCt method, with U6 as the internal control. **P < 0.01 versus controls.
Figure 3
 
Transforming growth factor-β1 enhances miR-200b expression. MicroRNA-200b levels were assessed by qRT-PCR and determined using the 2-ΔΔCt method, with U6 as the internal control. **P < 0.01 versus controls.
miR-200b Directly Targets and Inhibits CDKN1B and RND3/RhoE
We used the TargetScan 6.0, Diana microT 3.0, and miRanda algorithms to predict miR-200b target genes. From the large number of candidate target genes, we focused on CDKN1B and RND3. CDKN1B (p27/kip1) is a key regulator of cell cycle phase; it opposes cell cycle progression by inhibiting cyclin/CDK-complex activities in response to a variety of growth inhibitory signals as a negative regulator and is upregulated by TGF-β. 18,19,27 RND3 (RhoE) is an atypical RhoGTPase family member and is involved in the regulation of cell proliferation and cell cycle progression. 21,28 As shown in Figures 4A and 4B, the miR-200b target binding site regions are proposed to be 1584 to 1591 and 1729 to 1735 for RND3, and 1022 to 1027 for CDKN1B. To test whether miR-200b targets CDKN1B and RND3, we generated reporter constructs containing wild-type or mutated CDKN1B and RND3 3′-UTR sequences downstream of the luciferase coding sequence. As shown in Figures 3B and 4A, miR-200b significantly decreased luciferase activities in 293T cells transfected with the reporter constructs containing wild-type CDKN1B and RND3 3′-UTRs. However, miR-200b did not affect luciferase activity when cells were transfected with constructs containing no miR-200b. The luciferase activities in the mutated CDKN1B and RND3 3′-UTR reporter constructs demonstrated that the miR-200b target-binding site regions were 1022 to 1028 in CDKN1B and 1584 to 1591/1729 to 1735 in RND3. To further determine the regulation of CDKN1B and RND3 by miR-200b, HTFs were transfected with miR-200b mimics (100 nM), miR-200b inhibitors (200 nM), or their controls. Compared with untransfected cells or cells transfected with the scramble controls, miR-200b mimics suppressed the expression of p27/kip1 and RND3 at both the protein and mRNA levels (Figs. 4C, 4D). In previous studies, we indicated that treatment with TGF-β1 would cause an increase in miR-200b expression. To confirm the effect of miR-200b on p27/kip1 and RND3 expression and its signaling, we subjected HTFs to TGF-β1 treatment. Compared with control cells, treatment with TGF-β1 caused decreases in p27/kip1 and RND3 expression in HTFs. In addition, the effect of TGF-β1 on the down-regulated expression of p27/kip1 and RND3 was partially reversed by transfection of HTFs with the miR-200b inhibitor. p27/kip1 is the key negative regulator of cyclins E and A, and some reports have indicated that RND3 acts as a negative regulator of cyclin D. In contrast to the expression of p27/kip1 and RND3, enhanced expression of cyclins E and D1 was detected in HTFs. Together with the results from the 3′-UTR luciferase reporter study, we can conclude that miR-200b directly targets and inhibits p27/kip1 and RND3. 
Figure 4
 
Transforming growth factor-β1 directly targets CDKN1B (p27/kip1) and RND3 (RhoE). The predicted binding sites of miR-200b at the positions of 1584 to 1591 and 1729 to 1735 in the RhoE 3′UTR region and 1022 to 1028 in the p27 3′-UTR region are shown (yellow). The mutated sites are also shown (red) (A). The reduction of luciferase activity by RhoE and p27 3′UTR is dependent on miR-200b. The mutation of a predicted miR-200b binding site in both the RhoE and p27 3′UTRs abolished miR-200b–mediated repression of luciferase activity (B). After transfection with or without miR-200b mimics, inhibitors, or their controls, the HTFs were treated with or without 5.0 ng/mL TGF-β1 for 24 hours and then harvested. The mRNA of RhoE and p27/kip1 was detected by qRT-PCR and normalized to GAPDH mRNA levels (C). The expression of RhoE, p27/kip1, cyclin D1, and cyclin E was detected by Western blot analysis, and the expression of GAPDH was used as the loading control (D). *P < 0.05 versus controls. **P < 0.01 versus the control. # P < 0.05 versus TGF-β1 groups.
Figure 4
 
Transforming growth factor-β1 directly targets CDKN1B (p27/kip1) and RND3 (RhoE). The predicted binding sites of miR-200b at the positions of 1584 to 1591 and 1729 to 1735 in the RhoE 3′UTR region and 1022 to 1028 in the p27 3′-UTR region are shown (yellow). The mutated sites are also shown (red) (A). The reduction of luciferase activity by RhoE and p27 3′UTR is dependent on miR-200b. The mutation of a predicted miR-200b binding site in both the RhoE and p27 3′UTRs abolished miR-200b–mediated repression of luciferase activity (B). After transfection with or without miR-200b mimics, inhibitors, or their controls, the HTFs were treated with or without 5.0 ng/mL TGF-β1 for 24 hours and then harvested. The mRNA of RhoE and p27/kip1 was detected by qRT-PCR and normalized to GAPDH mRNA levels (C). The expression of RhoE, p27/kip1, cyclin D1, and cyclin E was detected by Western blot analysis, and the expression of GAPDH was used as the loading control (D). *P < 0.05 versus controls. **P < 0.01 versus the control. # P < 0.05 versus TGF-β1 groups.
Involvement of miR-200b in HTF Proliferation
To study the function of miR-200b in cell proliferation, HTFs were transfected with miR-200b mimics, miR-200b inhibitors, or their controls. As shown in Figure 5A, real-time PCR confirmed the increased expression of miR-200b in cells transfected with the miR-200b mimic and the decreased expression of miR-200b in cells transfected with the miR-200b inhibitors. Cell proliferation was measured using a CCK-8 assay kit. Compared with the untransfected cells or cells transfected with the scramble controls, miR-200b stimulated cell proliferation in HTF cells. The enhanced proliferation caused by the TGF-β1–induced upregulation of miR-200b was partially reversed by transfection of the HTFs with the miR-200b inhibitor (Fig. 5B). This result was also confirmed by the EdU incorporation assay and cell cycle analysis (Figs. 5C, 5D). All these results indicated a stimulatory function of miR-200b in the proliferation of TGF-β1–treated HTFs. 
Figure 5
 
MicroRNA-200b promotes HTF proliferation. After transfection with miR-200b mimics, inhibitors, or their controls, the levels of miR-200b in HTFs were detected by qRT-PCR. The results are presented as the fold-change compared with the controls (A). After treatment with or without 5.0 ng/mL TGF-β1 for 24 hours, HTFs were harvested to detected cell proliferation by the CCK-8 assay (B), cell cycle analysis (C), and Edu assay (100× magnification [D]). *P < 0.05 versus controls. **P < 0.01 versus controls. # P < 0.05 versus TGF-β1 groups.
Figure 5
 
MicroRNA-200b promotes HTF proliferation. After transfection with miR-200b mimics, inhibitors, or their controls, the levels of miR-200b in HTFs were detected by qRT-PCR. The results are presented as the fold-change compared with the controls (A). After treatment with or without 5.0 ng/mL TGF-β1 for 24 hours, HTFs were harvested to detected cell proliferation by the CCK-8 assay (B), cell cycle analysis (C), and Edu assay (100× magnification [D]). *P < 0.05 versus controls. **P < 0.01 versus controls. # P < 0.05 versus TGF-β1 groups.
Involvement of p27/kip1 and RND3 in the Proliferation of HTFs
To further elucidate the functions of p27/kip1 and RND3 in the proliferation of HTFs, we generated stable cell lines using lentivirus constructs expressing p27/kip1 and RND3 inhibitors or their scrambled controls. Western blot analysis confirmed the decreased expression levels of p27/kip1 and RND3 in the stable cell lines (Fig. 6A). Increased percentages of cells in S phase and increased expression of cyclin D1, cyclin E, and PCNA DNA were demonstrated in both p27/kip1 and RhoE knockdown cells (Figs. 6B, 6C). These results indicated that p27/kip1 and RND3 play a role in the proliferation of HTFs. 
Figure 6
 
p27/kip1 and RhoE are involved in HTF proliferation. The expression of p27/kip1 and RhoE in knockdown cells by the pLV-p27/kip1 and pLV-RhoE inhibitors was verified by Western blot analysis (A). The cell proliferation in HTFs was detected by cell cycle analysis (B) and the expression of cyclin D1, cyclin E, and PCNA (C). *P < 0.05 versus controls. ***P < 0.01 versus controls. The expression of GAPDH was used as the loading control.
Figure 6
 
p27/kip1 and RhoE are involved in HTF proliferation. The expression of p27/kip1 and RhoE in knockdown cells by the pLV-p27/kip1 and pLV-RhoE inhibitors was verified by Western blot analysis (A). The cell proliferation in HTFs was detected by cell cycle analysis (B) and the expression of cyclin D1, cyclin E, and PCNA (C). *P < 0.05 versus controls. ***P < 0.01 versus controls. The expression of GAPDH was used as the loading control.
Discussion
Transforming growth factor-β1 is a multifunctional growth factor and is known to be one of the most potent stimulating factors for the proliferation of fibroblasts. 29,30 For instance, TGF-β1–induced fibroblast proliferation has also been demonstrated in mouse embryonic fibroblasts, 31 rat kidney fibroblasts, 32,33 and mouse embryo–derived fibroblasts. 34 The plasma-driven TGF-β1 during glaucoma-filtering surgery, as well as the activated TGF-β1 expression in trabecular meshwork cells and optic nerve head astrocytes induced by high IOP, which subsequently stimulates the migration and proliferation of HTFs, leading to increased collagen production and ultimately resulting in subconjunctival fibrosis and scar formation. 7,35,36 The antifibrotic effects of anti–TGF-β molecules have stimulated interest in the prevention of scar formation following glaucoma-filtering surgery. TGF-β1 signal transduction involves the regulation of the expression of multiple genes by heterodimeric receptors I and II. 37 Several studies have demonstrated the useful effects of monoclonal antibodies, oligonucleotides, and RNA interference targeting TGF-β and TGF-β type II receptors in preventing scarring after trabeculectomy. 38,39 However, TGF-β–induced trans-differentiation of fibroblasts to myofibroblasts is at the core of the fibrotic process, which is essential for natural wound healing. 30 Completely blocking the effects of TGF-β or excessive suppression of the proliferation of fibroblasts with agents such as mitomycin C (MMC) and 5-fluorouracil (5-FU) may result in serious complications. 29,40 Thus, a new treatment strategy to modulate the excessive proliferation of fibroblasts is required. 
MicroRNA is a new approach for targeting TGF-β-induced HTF proliferation. In this study, miR-200b was upregulated by TGF-β treatment, similar to the results of the microarray study described by Li et al. 15 The five members of the human miR-200 family are located in two different loci in the genome and are divided into two clusters according to their chromosomal location: miR-200b, miR-200a, and miR-429 are located on chromosome 1p36, and miR-200c and miR-141 are located on chromosome 12p13. 41 MicroRNA-200b is highly associated with epithelial-mesenchymal transition (EMT) by targeting the E-box–binding transcription factors (ZEB1 and ZEB2) 42,43 and plays a key role in fibrosis in the lungs, 10 heart, 44 kidneys, 45 and stomach. 43 In contrast, Smad2, located downstream of the TGF-β type II receptor, has been reported to be the target gene for miRNA-200b in the promotion of intestinal epithelial cell proliferation, and miR-200b inhibits cyclinD1 expression by targeting GATA-4 to inhibit cardiac myocyte proliferation. 44,46  
In this study, increased miR-200b (by TGF-β1 treatment or transient overexpression) significantly promoted the proliferation of HTFs, and this effect could be blocked by the inhibitor of miR200b. These results indicate that TGF-β1 stimulates the proliferation of HTFs via miR-200b. The luciferase activity assays indicate that p27/kip1 and RND3 (RhoE) were the direct targets of miR-200b, which was further confirmed in assays of miR-200b–mediated p27/kip1 and RhoE expression. Therefore, when miR-200b expression was high, the expression levels of p27/kip1 and RND3 (RhoE) were low and vice versa. p27/kip1, as a negative cell cycle regulator, acts in G0 and G1 to inhibit complexes of G1 cyclins and cyclin-dependent kinases (CDKs), especially cyclin E-Cdk2 complexes. 1820 In this study, the knockdown of p27/kip1 in HTFs resulted in increased cell proliferation and cyclin E expression. RhoE was also reported to regulate proliferation and survival in some human tumor cell lines. RhoE is a member of the Rho GTPase family and is involved in the organization of the actin cytoskeleton and the regulation of cell cycle progression by reducing the cell cycle regulators, including cyclin D1. In contrast, the knockdown of RhoE in HTFs also increased HTF proliferation and cyclin D1 expression, indicating its role in the cell cycle. To the best of our knowledge, this is the first report demonstrating the role of RhoE in the proliferation of fibroblasts. 
In conclusion, this study provides some novel findings with regard to HTFs. First, miR-200b expression was found to be a signature of the response of HTFs to TGF-β1 exposure. Second, the findings of this study first established a link between the proliferation of HTFs and miR-200b in vitro. Finally, this is the first study to describe p27/kip1 as a direct target of miR-200b. Our findings also suggest that miR-200b may be a good therapeutic target for controlling postoperative subconjunctival fibrosis following glaucoma-filtering surgery. 
Acknowledgments
Supported in part by the National Natural Science Foundation of China (Grant 81271001). 
Disclosure: J. Tong, None; Y. Fu, None; X. Xu, None; S. Fan, None; H. Sun, None; Y. Liang, None; K. Xu, None; Z. Yuan, None; Y. Ge, None 
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Figure 1
 
Fibroblasts in identification. Cells migrated from the adherent human Tenon's tissue after 4 to 7 days and cells confluenced 90% or more after passages. (A) Pictures were taken at 100× magnification. Human Tenon's fibroblasts cells were incubated with vimentin, cytokeratin, and fibroblast surface protein antibodies. (B) Immunofluorescence with vimentin-positive staining (green), and nuclear staining with DAPI (blue). (C) Immunofluorescence with FSP-positive staining (red), and nuclear staining with DAPI (blue). (D) Negative cytokeratin staining (green), and nuclei labeled with DAPI (blue). All pictures were taken at 400× magnification.
Figure 1
 
Fibroblasts in identification. Cells migrated from the adherent human Tenon's tissue after 4 to 7 days and cells confluenced 90% or more after passages. (A) Pictures were taken at 100× magnification. Human Tenon's fibroblasts cells were incubated with vimentin, cytokeratin, and fibroblast surface protein antibodies. (B) Immunofluorescence with vimentin-positive staining (green), and nuclear staining with DAPI (blue). (C) Immunofluorescence with FSP-positive staining (red), and nuclear staining with DAPI (blue). (D) Negative cytokeratin staining (green), and nuclei labeled with DAPI (blue). All pictures were taken at 400× magnification.
Figure 2
 
Transforming growth factor-β1 promotes HTF proliferation. Human Tenon's capsule fibroblasts were treated with TGF-β1 for 24 hours at doses of 0, 0.5, 1.0, 2.0, 5.0, and 10 ng/mL, and cell proliferation was detected using the CCK-8 assay (A) and cell cycle analysis (B). Human Tenon's capsule fibroblasts were treated with TGF-β1 at a dose of 5.0 ng/mL for 0, 12, 24, and 48 hours, and cell proliferation was detected using the CCK-8 assay (C) and by assessing the expression of cell proliferation markers (cyclin D1, cyclin E, and PCNA [D]). *P < 0.05 versus controls. **P < 0.01 versus controls.
Figure 2
 
Transforming growth factor-β1 promotes HTF proliferation. Human Tenon's capsule fibroblasts were treated with TGF-β1 for 24 hours at doses of 0, 0.5, 1.0, 2.0, 5.0, and 10 ng/mL, and cell proliferation was detected using the CCK-8 assay (A) and cell cycle analysis (B). Human Tenon's capsule fibroblasts were treated with TGF-β1 at a dose of 5.0 ng/mL for 0, 12, 24, and 48 hours, and cell proliferation was detected using the CCK-8 assay (C) and by assessing the expression of cell proliferation markers (cyclin D1, cyclin E, and PCNA [D]). *P < 0.05 versus controls. **P < 0.01 versus controls.
Figure 3
 
Transforming growth factor-β1 enhances miR-200b expression. MicroRNA-200b levels were assessed by qRT-PCR and determined using the 2-ΔΔCt method, with U6 as the internal control. **P < 0.01 versus controls.
Figure 3
 
Transforming growth factor-β1 enhances miR-200b expression. MicroRNA-200b levels were assessed by qRT-PCR and determined using the 2-ΔΔCt method, with U6 as the internal control. **P < 0.01 versus controls.
Figure 4
 
Transforming growth factor-β1 directly targets CDKN1B (p27/kip1) and RND3 (RhoE). The predicted binding sites of miR-200b at the positions of 1584 to 1591 and 1729 to 1735 in the RhoE 3′UTR region and 1022 to 1028 in the p27 3′-UTR region are shown (yellow). The mutated sites are also shown (red) (A). The reduction of luciferase activity by RhoE and p27 3′UTR is dependent on miR-200b. The mutation of a predicted miR-200b binding site in both the RhoE and p27 3′UTRs abolished miR-200b–mediated repression of luciferase activity (B). After transfection with or without miR-200b mimics, inhibitors, or their controls, the HTFs were treated with or without 5.0 ng/mL TGF-β1 for 24 hours and then harvested. The mRNA of RhoE and p27/kip1 was detected by qRT-PCR and normalized to GAPDH mRNA levels (C). The expression of RhoE, p27/kip1, cyclin D1, and cyclin E was detected by Western blot analysis, and the expression of GAPDH was used as the loading control (D). *P < 0.05 versus controls. **P < 0.01 versus the control. # P < 0.05 versus TGF-β1 groups.
Figure 4
 
Transforming growth factor-β1 directly targets CDKN1B (p27/kip1) and RND3 (RhoE). The predicted binding sites of miR-200b at the positions of 1584 to 1591 and 1729 to 1735 in the RhoE 3′UTR region and 1022 to 1028 in the p27 3′-UTR region are shown (yellow). The mutated sites are also shown (red) (A). The reduction of luciferase activity by RhoE and p27 3′UTR is dependent on miR-200b. The mutation of a predicted miR-200b binding site in both the RhoE and p27 3′UTRs abolished miR-200b–mediated repression of luciferase activity (B). After transfection with or without miR-200b mimics, inhibitors, or their controls, the HTFs were treated with or without 5.0 ng/mL TGF-β1 for 24 hours and then harvested. The mRNA of RhoE and p27/kip1 was detected by qRT-PCR and normalized to GAPDH mRNA levels (C). The expression of RhoE, p27/kip1, cyclin D1, and cyclin E was detected by Western blot analysis, and the expression of GAPDH was used as the loading control (D). *P < 0.05 versus controls. **P < 0.01 versus the control. # P < 0.05 versus TGF-β1 groups.
Figure 5
 
MicroRNA-200b promotes HTF proliferation. After transfection with miR-200b mimics, inhibitors, or their controls, the levels of miR-200b in HTFs were detected by qRT-PCR. The results are presented as the fold-change compared with the controls (A). After treatment with or without 5.0 ng/mL TGF-β1 for 24 hours, HTFs were harvested to detected cell proliferation by the CCK-8 assay (B), cell cycle analysis (C), and Edu assay (100× magnification [D]). *P < 0.05 versus controls. **P < 0.01 versus controls. # P < 0.05 versus TGF-β1 groups.
Figure 5
 
MicroRNA-200b promotes HTF proliferation. After transfection with miR-200b mimics, inhibitors, or their controls, the levels of miR-200b in HTFs were detected by qRT-PCR. The results are presented as the fold-change compared with the controls (A). After treatment with or without 5.0 ng/mL TGF-β1 for 24 hours, HTFs were harvested to detected cell proliferation by the CCK-8 assay (B), cell cycle analysis (C), and Edu assay (100× magnification [D]). *P < 0.05 versus controls. **P < 0.01 versus controls. # P < 0.05 versus TGF-β1 groups.
Figure 6
 
p27/kip1 and RhoE are involved in HTF proliferation. The expression of p27/kip1 and RhoE in knockdown cells by the pLV-p27/kip1 and pLV-RhoE inhibitors was verified by Western blot analysis (A). The cell proliferation in HTFs was detected by cell cycle analysis (B) and the expression of cyclin D1, cyclin E, and PCNA (C). *P < 0.05 versus controls. ***P < 0.01 versus controls. The expression of GAPDH was used as the loading control.
Figure 6
 
p27/kip1 and RhoE are involved in HTF proliferation. The expression of p27/kip1 and RhoE in knockdown cells by the pLV-p27/kip1 and pLV-RhoE inhibitors was verified by Western blot analysis (A). The cell proliferation in HTFs was detected by cell cycle analysis (B) and the expression of cyclin D1, cyclin E, and PCNA (C). *P < 0.05 versus controls. ***P < 0.01 versus controls. The expression of GAPDH was used as the loading control.
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