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Downregulation of miR-18a induces CTGF and promotes proliferation and migration of sodium hyaluronate treated human corneal epithelial cells
Downregulation of miR-18a induces CTGF and promotes proliferation and migration of sodium hyaluronate treated human corneal epithelial cells Yingzhuo Guo, Xiaohe Lu, Hua Wang PII: DOI: Reference:
S0378-1119(16)30534-0 doi: 10.1016/j.gene.2016.07.008 GENE 41441
To appear in:
Gene
Received date: Revised date: Accepted date:
10 January 2016 12 May 2016 3 July 2016
Please cite this article as: Guo, Yingzhuo, Lu, Xiaohe, Wang, Hua, Downregulation of miR-18a induces CTGF and promotes proliferation and migration of sodium hyaluronate treated human corneal epithelial cells, Gene (2016), doi: 10.1016/j.gene.2016.07.008
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ACCEPTED MANUSCRIPT Downregulation of miR-18a induces CTGF and promotes proliferation and migration of sodium hyaluronate treated human corneal epithelial
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cells
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Yingzhuo Guoa,b, Xiaohe Lua*, Hua Wangb
Department of Ophthalmology, Zhujiang Hospital, Southern Medical University, Guangzhou
510280 , Guangdong Province, China
Department of Ophthalmology& Optometry, Hunan Provincial People's Hospital, Changsha,
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b
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410005, Hunan Province,China.
*Corresponding author: Xiaohe Lu, Department of Ophthalmology, Zhujiang Hospital,
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Southern Medical University, No. 253 Industrial Road，Guangzhou 510280 , Guangdong
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Province, China (Email: [email protected] )
ACCEPTED MANUSCRIPT ABSTRACT
Properly controlled corneal epithelial wound healing is critical for health of
cornea, which involves cell proliferation, migration, anchoring and differentiation. Sodium
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hyaluronate (SH) has been proven to exert beneficial pharmacological effect on corneal wound
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healing, though the underlying mechanism remained open to investigation. MicroRNAs (miRNAs) are small single-stranded RNAs that could bind to 3’UTR of mRNAs of target genes. The multi-target regulation of miRNAs may favor treatment of corneal wound given the
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complicated processes implicated in the healing process, which has inspired initiatives to
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develop miRNA therapy in corneal wound healing. In this light, we used miRNAs profiling to detect whether miRNAs are also implicated in the mechanism underlying the stimulatory effect
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of SH on corneal epithelial wound healing. We found miR-18a was most susceptible to SH
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treatment, the target prediction of which were enriched in a bunch of pathways implicated in
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corneal wound healing. Connective tissue growth factor (CTGF) was found to be overrepresented in most significant enriched pathways and was experimentally confirmed as a
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bona fide target of miR-18a, which modulated cell migration and proliferation of human corneal epithelial cells.
Keywords: corneal epithelial wound healing; sodium hyaluronate; miR-18a; CTGF
ACCEPTED MANUSCRIPT INTROCUTION Corneal wound healing encompasses a sequential and highly coordinated events, including cell death, proliferation, migration, differentiation and extracellular matrix remodeling[1]. The
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corneal epithelium is the outermost part of the ocular surface, where the epithelial cells are
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constantly regenerating to maintain a clear and proper functioning cornea. Corneal epithelial
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wounds can be caused by mechanical abrasion, chemical burn or ophthalmic surgeries, which, if not adequately healed, can result in corneal haze, ulcer, perforations, refractory epithelial defect or blindness[2]. Therefore, rapid and proper regeneration of epithelium is of vital
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importance for corneal wound healing. Epithelial healing at the corneal surface is a dynamic process that involves proliferation of basal epithelial cells, centripetal and circumferential
underneath connective tissue[3].
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migration of epithelial cell sheets, and anchoring of newly generated epithelium to the
Sodium hyaluronate (SH) is a naturally occurring polysaccharide of high viscoelasticity, which
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has been used in ophthalmic surgery to protect the corneal epithelium and endothelium[4]. It
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has also been incorporated into artificial tears to treat dry eye[5]. Studies of animal corneal
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wound model showed that SH could stimulate cell migration, adhesion, and proliferation during corneal epithelial wound healing[6], which was found to be peculiar for SH since other polysaccharides, such as chondroitin sulphate, keratan sulphate and heparan sulphate, could
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not produce similar effect. This indicates that the therapeutic effect of SH on corneal epithelial wound healing is not only dependent on its viscoelasticity, but also pharmacological factors[7], though the underlying mechanism remained controversial[3]. MicroRNAs are 22-24 nt non-coding single-stranded RNAs that can bind to sequences on 3’UTR of target mRNAs, which lead to perturbed translation or degradation of target mRNAs[8]–[10]. Over a decade of exploration, miRNAs are proved to be powerful gene expression regulators that mediate various biological processes[11], [12]. The importance of miRNAs in corneal wound healing has been highlighted by the evidence that some miRNAs promote healing but others inhibit it[9], [13]–[16]. Due to the miRNA-miRNA variability, they can usually target multiple genes[17]. Since wound healing is a complex process, the multi-target regulation of miRNAs may favor treatment that requires combinatorial solutions. Indeed, the application of anntagomir-based therapy has been performed in correcting the aberrance of
ACCEPTED MANUSCRIPT epithelial wound healing in human diabetic organ-culture corneas[13], [16]. Recently, Lilly et al. reported
hyaluronan treatment could promote production
of
miR-10b,
leading to
Rho-kinase-associated breast tumor cell invasion[18]. This prompted us to investigate
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changes of the miRNA profile in corneal epithelial cells (HCECs) in response to SH, in an attempt to address the mechanism underlying the accelerated wound healing process.
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In this study, we demonstrated subtle alteration in miRNAs profile in HCECs following SH treatment, and identified miR-18a as most susceptible to treatment, which was substantially downregulated. Subsequent target prediction combined with functional enrichment analysis
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identified a few pathways that are possiblely targeted by miR-18a, which offers candidates for further investigation. In “Arf6 downstream pathway” that ranked at the top of predictions, we
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verified CTGF as a functional target in wound healing, the knock-down of which was found to significantly inhibit proliferation and migration of HCECs.
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MATERIAL AND METHODS
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Cell culture and transfection with miR-18a antagomir, mimic and CTGF siRNA Human corneal epithelial cells (ThermoFisher) were maintained in human keratinocyte growth
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medium (KGM-2; Invitrogen, CA) and incubated at 37℃, humid air containing 5% CO2. Cells were then seeded onto 96-well plates and grown to about 80–90% confluence. The morphology of cultures were evaluated on daily basis by phase contrast microscopy. Three 2+
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days before experiment, cells were transferred to KGM-2 supplemented with 1mM Ca for 12h, and then washed with sterile phosphate-buffered saline (PBS). At day four, cell migration was evaluated and six similar cultures were chosen and randomly assigned to control or experimental group, each containing three biological replicates. Control group and experimental group were cultured in KGM-2 alone and KGM-2 with SH (0.6mg/ml), respectively. Transfection Scrambled control or miR-18a (or miR-92) antagomir/mimic (ThermoFisher, Ambion) were introduced into HCECs using Lipofectamine RNAi Max (ThermoFisher) for 6 h. Cells were harvested for assays two days after transfection. For CTGF siRNA transfection, lentivirus expressing CTGF siRNA was constructed and amplified as described by Wang et al.[19].
ACCEPTED MANUSCRIPT Pooled lentiviral siRNA and scrambled siRNA (Scr-siRNA) were prepared for targeting human CTGF and as negative control, respectively. MiRNA microarray analysis
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Agilent 2100 Bioanalyzer was used to assess RNA quality. Finally 100 ng of total RNA was processed for use on the microarray using the FlashTagTM HSR labeling kit (Genisphere LLC)
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as per manufacturer’s protocols, in which the cRNA was generated and biotinylated to hybridized to the GeneChip miRNA Array (Affymetrix). Affymetrix Model 3000 scanner wasused to scan to array accordingly. MiRNA QC tool software (Affymetrix) was used to
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generaate expression value which was normalized and log transformed. Differential expression was detected by R package limma, which was defined as log2 fold change>=1.5 or
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<=-1.5 (adjP<0.05).
Target Analysis and pathway common analysis
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TargetScan Release 7.0 (http://www.targetscan.org/) was employed to predict targets of
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miR-18a, which is based on searching for the presence of 8mer, 7mer, and 6mer sites that match the seed region of each miRNA. The 2,898 predictions were ranked by “cumulative
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weighted context ++ score”, and top 200 were selected for pathway common analysis to identify functional clusters that are of biological significance related to miR-18a and may contain target genes. Webgestalt[20], an online webserver for functional enrichment analysis,
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was adopted to perform pathway common enrichment using hypergeometric test. Pathway analysis was initially used to identify significant pathways linkage to differentially expressed genes. Likewise, as miR-18a may target multiple genes, the overrepresented pathway for prediction may address biological implication of miR-18a, within which the target will be included. The minimum genes contained in each pathway was set to “2” and significance “p=0.05” corrected by Benjamini-Hochberg (FDR) method. Quantitative RT-PCR TRIzol Reagent (Invitrogen) was used to extract total RNA from HCECs, the quality of which was confirmed. Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA from 10 ng total RNA. The expression of miRNAs or mRNA (CTGF) was quantified by RT-PCR using 7500 Fast Real-Time PCR System (Applied Biosystems), which was then normalized to U6B small nuclear RNA (RNU6B) or GAPDH depending on the
ACCEPTED MANUSCRIPT experiment. Cell viability assay The number of viable cells was determined by Methylthiazolyldiphenyl-tetrazolium bromide
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assay (Sigma-Aldrich). Cells were washed with PBS to remove non-adherent cells, and plated
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at 5,000 per cell, which was incubated for 6 hours. MTT reagent was added to the well, 10μL
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per well. Incubate the wells for 3 hrs at 37℃ until purple precipitate was visible. The cells were then added with 100μL detergent reagent per well and placed in the dark for 2 hrs, after which the absorbance at 570nm were taken.
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Western blot analysis
Cells were lysed in NP-40 buffer containing 150mM NaCl, 1.0% NP-40, 50mM Tris-HCl and
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protease inhibitors (Roche) and 1mM phenylmethyl sulfonylfluoride for 30 minutes on ice. The supernatant was subject to protein denaturation with buffer containing 2% sodium
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dodecylsulfate (SDS; Sigma), was subsequently analyzed using SDS-PAGE and monoclona
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antibody against CTGF. The resultant gel was then washed and covered by appropriate horseradish peroxidase-conjugated Ig secondary antibodies. Enhanced chemiluminescence
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(GE Healthcare) was used to detect the staining. Luciferase activity assay
CTGF 3’UTR and its mutated counterpart sequence plasmids were constructed. Cells were 5
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seeded in a 24-well plate at cell density of 1×10 cells/well. After 24 hours of culture, cells were cotransfected with firefly luciferase reporter plasmids respectively containing wild-type or mutant CTGF 3’UTR pRL-TK vector expressing Renilla luciferase, and miR-18a or miRNA negative control via Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured after 36 hours by Dual Luciferase Reporter Assay (Promega, US). Each tranfection was performed twice in triplicate.
Scratch wound healing assay Twenty-four hours after transfection with scrambled control antagomir/mimic (100 nmol/L), miR-18a antagomir/mimic, or CTGF siRNA, and a 200μL pipette tip was used to make a straight scratch on the culture, followed by washing with Hanks medium until no floating cells were present. The scratched cultures were then maintained in serum free medium.
ACCEPTED MANUSCRIPT Photographs were taken immediately and at 24h after scratches were made. Statistical analysis Results are expressed as mean values±std., and a Student’s t-test was used for testing
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statistical significance. P<0.05 is considered as significant, whereas P<0.001 represents extreme significance.
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RESULTS
Sodium hyaluronate (SH) modulates expression of microRNA in corneal epithelial cells
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Microarray was conducted to profile the microRNA expression in human corneal epithelial cells (HCECs) after 2 days exposure to 0.6mg/ml SH[21]. Differentially expressed microRNAs were There was only a few
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defined as those with log2 fold change>=1.5 or <=-1.5 (adjP<0.05).
significant variation in treated cells compared with control (Figure 1A). In total, 16 miRNAs were deregulated, among which miR-18a and miR-92 were most highly downregulated, and
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miR-210, miR-26b, and miR-378 showed the largest fold change. The number of induced
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miRNAs and their repressed counterparts were similar, however, the repressed miRNAs showed higher absolute fold change, implying that one or more particular biological processes
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may be activated, to mediate control of corneal wound healing. We then performed RT-PCR to confirm the differentially expressed miRNAs. As anticipated, miR-18a was dramatically repressed (logFC (log-transformed fold change) = -1.987983, p=2.05E-13; Figure 1B), next to
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which was miR-92 (logFC=-1.228615, p=6.52E-12; Figure 1B).
Antagomir and mimics of miR-18a respectively enhanced and deterred the proliferation of HCECs Since miR-18a showed predominantly large absolute fold change among all deregulated miRNAs, we reasoned that miR-18a may be essential for promoting wound healing in SH treated cells. To examine the critical role of miR-18a in corneal wound healing, we used mimics and antagomir to transfect HCECs to see if the cell proliferation will be regulated accordingly. The transfection efficiency was confirmed by quantifying miR-18a for every treatment or transfection (Figure 1C). The cell viability was measured for 3 days to monitor the cell growth. The HCECs transfected with miR-18a mimics showed cell growth rate slower than
ACCEPTED MANUSCRIPT HCECs transfected with scramble sequences,
while those transfected with miR-18a
antagomir displayed accelerated growth rate. (Figure 1D). Putative targets of miR-18a were enriched in Arf6 downstream pathway
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Since miRNAs function by mediating partial or entire degradation of mRNAs of target genes, determination of target genes are critical for elucidating the role of miR-18a in HCECs. To
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address this, we adopted Targetscan, a tool based on searching for the presence of 8mer, 7mer, and 6mer sites that match the seed region of each miRNA, to predict target genes of miR-18a. There were 2, 898 transcripts predicted as target of miR-18a, which were
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ranked by “cumulative weighted context ++ score”, a measure indicating the confidence of prediction (data not shown).
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We then adopted enrichment analysis to identify functional clusters containing genes that mediate re-epithelialization of HCECs. To narrow down the gene set, top 200 genes were
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selected for pathway common analysis ( the list is available at : http://www.targetscan.org)[20].
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These predicted genes were enriched in multiple pathways implicated in cell proliferation, cell cycle, and cell mobility (Table 1). These pathways include “Arf6 downstream pathway”, “VEGF
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and VEGFR signaling network”, “Nectin adhesion pathway”, “Arf6 trafficking events”, “Endothelins”, and “E-cadherin signaling in the nascent adherens junctionn”. In particular, we noticed that “Arf6 downstream pathway“ and “Arf6 trafficking events” are both involved in the
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formation of lamellipodia at the leading edge of migrating corneal epithelial cells and in the growing cones of corneal nerve fibers[22]. This agreement validates the roles of these pathways in corneal wound healing. “Arf6 downstream pathway” contained 22 putative target genes, including components of cell division cycle, such as CDC42 (cell division cycle 42) and CCND2 (cyclin D2), and of cell growth, such as FRS2 (fibroblast growth factor receptor substrate 2) and CTGF (connective tissue growth factor). CTGF is a target of miR-18a CDC42 and CCND2 are critical component of cell cycle and cell proliferation, and are ubiquitous in various cell types[11], [23], [24]. We presented that miR-18a mediated both cell proliferation, therefore, we reasoned that CDC42 and CCND2 may not be the major factors that control corneal wound healing after treatment of SH, since migration entails not only cell proliferation but also cell motility, such as cell-to-cell communication. Both FRS2 and CTGF
ACCEPTED MANUSCRIPT were reported to be associated with cell migration[25]–[28]. Recently, Blalock et al. revealed that expression of CTGF could mediate effect of TGF-beta induction of COLA1A synthesis in corneal fibroblasts, which promotes corneal wound healing[29]. In this light, we postulated that
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miR-18a might modulate biological processes by suppressing CTGF. To testify our speculation, we performed both quantitative RT-PCR and western blotting to detect CTGF expression in
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HCECs after being transfected with miR-18a mimics and antagomir, respectively. Transfection with miR-18a antagomir resulted in a remarkable increase in CTGF protein, while the HCECs transfected with miR-18a mimics present substantial reduction (Figure 2A, B). In addition, the
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level of CTGF in SH treated cells was dramatically elevated compared to untreated cells (scrambled control), suggesting that CTGF is likely a bona fide target of miR-18a and may
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respond to SH treatment.
In addition, we further conducted dual luciferase reporter assay to verify whether CTGF is a
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bona fide target of miR-18a. The CTGF mRNA 3’ UTR or CTGF 3’UTR with mutation
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sequence plasmids (Figure 2C) were cotransfected with miR-18a or negative control into HCECs. After 36 hours, the luciferase activity was measured. The result showed that HCEC
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cells transfected with miR-18a mimics had luciferase activity significantly lower than control, suggesting that CTGF is a target of miR-18a (Figure 2D). Silencing of CTGF counteracted the migration and proliferation enhanced by SH
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Although we demonstrated that CTGF is the target of miR-18a in HCECs, as miRNAs usually target multiple genes, the role of CTGF in SH treated HCECs remained to be determined. We measured the changes in cell viability and scratch wound healing rate of CTGF silenced HCECs treated with SH. Knockdown efficiency was confirmed at mRNA and protein level (Figure 3A, 3B). The control demonstrated significantly higher growth rate during observation, while the CTGF silenced cells display slow growth during the first two days of observation (Figure 3C, 3D). As anticipated, in wound healing, CTGF ablated HCECs showed impaired cell mobility (Figure 3E, 3F), which is corresponding to protein level of CTGF (Figure 3A). Impairment of proliferation and migration resulting from CTGF deficiency indicates the critical role of CTGF in the underlying mechanism. DISCUSSION
ACCEPTED MANUSCRIPT The regulatory role of miRNAs had been well documented in the studies of eye development and pathology. Mutations of dicer, a pivotal enzyme responsible for cleaving double-stranded RNA into double stranded pre-miRNA, could result in defects in normal development of retina,
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lens, and cornea[30]–[32]. In addition to global aberrance in regulation of miRNA due to deficient dicer, specific miRNAs have also been identified as functional in corneal epithelial
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development, which indicates their therapeutic potentials. For example, miR-145 modulates the corneal epithelium formation and maintenance of epithelial integrity via targeting ITGB8[33]. miR-31, which is preferentially expressed in epithelial, downregulates factor
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inhibiting hypoxia-inducible factor-1 (FIH-1) and thus affects glycogen metabolism and keratinocyte differentiation in corneal epithelial cells[34], [35]. These discoveries highlight the
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importance of miRNAs in corneal epithelial homeostasis. Indeed, the miRNAs expression display a tissue specific pattern in cornea. miR-184 is highly expressed in epithelium of mouse
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lens and central cornea[36]; miR-124 and miR-204 were concordantly expressed in all lens
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epithelial cells, while miR-205 was expressed all over the corneal epithelium[37]. In addition, the short length of miRNAs preclude its one-on-one targeting to respective target genes, which
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means that a miRNA could target multiple genes and vice versa. Therefore, a holistic scanning of miRNAs would facilitate identification of effector miRNAs. The nuance difference between miRNA profiles of SH treated cells and control attested to this necessity, since the few
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differentially expressed miRNAs might be missed without complete profiling. Corneal epithelial healing involves highly orchestrated events including cell proliferation, migration, adhesion and differentiation in order to maintain the integrity and health of corneal epithelial surface[38]. The application of SH in treating corneal wound healing is well-established. Hyaluronic acid and high potassium ion concentration artificial tears could promote corneal epithelial wound healing in scraping model[39], and the stimulatory effect of HA on migration of rabbit corneal epithelial cells in vitro has been documented[40]. Recently, miRNA was highlighted as one of the novel technologies developed to manipulate corneal wound healing. In this light, we performed microarray to detect deregulation of miRNAs in response to SH treatment in HCECs, in an attempt to uncover the potential role of miRNAs in physiological mechanism underlying the enhancement of wound healing by SH. The concentration of 0.6mg/ml was used in the present study in keeping with previous studies,
ACCEPTED MANUSCRIPT which has been through gradient testing of optimal concentrations between 0.4~1.0mg/ml[3]. The largely consistent expression level of the majority of miRNAs between SH treated cells and control implied the effect and safety of using SH in treating corneal epithelial injury[3],
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since it’s likely that the proliferation and migration were promoted at the expense of less perturbation in other biological pathways.
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Among the virtually maintained miRNAs, miR-18a and miR-92 exhibited prominent downregulation, which was then verified by RT-qPCR where miR-18a showed predominantly lower fold change. This contrast projects the major regulatory role of miR-18a under SH
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treatment, and the inhibitory function of miR-18a was evidenced by the observation of deterred
transfected with miR-18a antagomir.
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proliferation in cells transfected with miR-18a mimics and enhanced proliferation in cells
Target gene prediction of miR-18a by TargetScan renders over two thousand hits, from which
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we selected top 200 predictions of context++ model. This model was more predictive than any
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published model and even equivalent with in vivo crosslinking approaches in miRNA target prediction[41]. Pathwaycommon enrichment was employed to infer functional clusters that
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may contain target genes. We arrived at six pathways highly implicated in cell migration and proliferation, including “Arf6 downstream pathway”, “VEGF and VEGFR signaling network”, “Nectin adhesion pathway”, “Arf6 trafficking events”, “Endothelins”, and “E-cadherin signaling
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in the nascent adherens junctionn”, among which “Arf6 downstream pathway” ranked at the top. Vascular endothelial growth factor (VEGF), was originally identified as a growth factor in angiogenesis. However, it has also been implicated in mediating cell proliferation in various epidermal tumors. Autocrine VEGF signaling synergizing with EGFR in tumor cells has the ability to promote epithelial cancer development[42]. Recently, several studies demonstrated the stimulatory effect of VEGF on multiple biological processes of the wound healing cascade, including vascularization, epithelization and collagen deposition[43]. Therefore, it is reasonable to postulate a potential similar role of VEGF in corneal epithelium. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, was found in human corneal epithelium, and more interestingly, in rabbit tear fluid[44]. Hitoshi et al. highlighted the therapeutic value of ET-1 in remedy of corneal epithelial defects by demonstrating elevated wound healing rate in culture corneal epithelial cells treated with ET-1 containing eyedrops[45]. Cell proliferation is critical for
ACCEPTED MANUSCRIPT continuous renewal of cornea and recovery from corneal trauma and keratoplasty, while during corneal epithelial wound healing, the progenitor corneal epithelial cells at the periphery may undergo formation of lamellipodia, protrusions that favor exploration of local environment and
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cognate cells, followed by epithelial to mesenchymal transition (EMT), a hallmark feature in the process of tumor invasion and wound healing[46], [47]. Both nectin and cadherin-based
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cell-cell adhesions are play important roles in adherens junctions of epithelial cells[48]. Whether the nascent junction between cells will grow and mature or decompose to promote proliferation is determined by Rac1 activity[49]. Transient presence of Rac1 is essential for
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membrane exploration at nasceent junctions, but thereafter, it would deter the maturation and undermine the junction. Felipe et al. revealed that during epithelial cell scattering, a process
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characteristic of EMT, cell-cell dissociation could be induced by initial decrease of Rac1, and this downregulation is ARF6-dependent[50]. Although common pathway enrichment was
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utilized to pin-point functional modules that may contain target genes of miR-18a, considering
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the large average number of miRNA targets, we cannot rule out that a subset of predictions may be bona fide targets. In addition, the engagment of these overrepresented pathways in
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the regulatory netwok of wound healing reinforces this possibility. Arf6 signaling was deemed to be most pertinent to epithelialization among the enriched pathways. Of the 22 genes contained in “Arf6 downstream signaling”, we chose CTGF as
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candidate, which was verified experimentally. Indeed, a previous study reported that miR-18a has a functional site in the CTGF 3’-UTR, and its etopic expression dramatically diminished CTGF mRNA levels as well as proteins[51], which was correlated with survival in glioblastoma. This agreement suggests that corneal wound healing and cancer metastasis share common biological processes. CTGF was identified as an important component in corneal wound healing and scar formation[52]. The role of CTGF in corneal wound healing was further evidenced by the nullification of CTGF deficiency on cell mobility proved to be stimulated by SH, which is consistent with a recent study by Daniel and colleagues where they observed immediate upregulation of CTGF in the epithelium at the wound margin and maintained high expression of CTGF during re-epithelialization[53]. They also reported a 40% reduction in the corneal re-epithelialization rate in conditional CTGF knock-out mouse model. Similarly, Lee et al. found that CTGF expression was higher in synovial cells deprived of serum but treated with
ACCEPTED MANUSCRIPT higher concentration of hyaluronic acid[54]. Although the molecular processes elicited by increase of CTGF which were conducive to epithelialization remained to be delineated, our study uncover a mechanism whereby SH demonstrated its therapeutic value in corneal
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epithelial wound healing, which implicates reduction of miR-18a and increase of CTGF therefrom. In addition, the observation that HA could expidite the synthesis of ECM and the cell
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could bind with CD44, the endogeneous receptor of HA, to facilitate cell migration[54], offers an alternative explanation for the stimulatory effect of SA on corneal epithelial wound healing. Recently, Yuchen et al. found that downregulation of CD44 inhibited transcription of YAP
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downstream effectors CTGF, Cyr61 and EDN1[55]. In the present study, the miR-18a mimics cannot completely block the expression of CTGF, indicating another mechanism controling
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expression of CTGF, which may involve CD44. Taken together, our study provides a complementary explanation for the stimulatory effect of SH on corneal epithelial wound
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healing.
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In summary, we observed subtle alterations in miRNA profile in HCECs following SH treatment, and miR-18a stood out as most susceptible. Target prediction yields an enormous amount of
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candidates, which was narrowed down by functional enrichment analysis. The six overrepresented pathways are highly implicated in multiple components of corneal epithelial wound healing, such as cell proliferation, formation of lamellipodia, and EMT, suggesting that
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multiple genes in those pathways may be bona fide targets of miR-18a, though we finally fixed on CTGF which was verified experimentally. Although the underlying mechanism remained to be delineated, our study revealed its role in the therapeutic effect of SH on corneal epithelial wound healing in relation to reduction of miR-18a. In addition, the functional clusters identified based on predicted targets of miR-18a may offer candidates for futher exploration. CONFLICT OF INTEREST None.
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LEGENDS Figure 1A. Differentially expressed miRNAs between SH treated cells and control.
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Volcano plot of miRNA profile. Red dots represent miRNAs are were detected as significantly
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expressed by p-value, and green dots represent non-siginificantly expressed miRNAs. y-axis
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denotes negative log10 transformed p-values, and x-axis present log2 transformed fold change. The differential expressed miRNAs in our study were defined as those with significant log2 fold change>=1.5 or <=-1.5.
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Figure 1B. Quantitative RT-PCR verification of miR-18a and miR-92 against control. miR-18a and miR-92 were most dramatically downregulated. y-axis indicates fold change
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against control, and x-axis denotes the expression level of miR-18a or miR-92 in SH treated cells. Error bar indicates standard errors. p-value indicates the significance of difference by
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Student’s test.
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Figure 1C. Expression of miR-18a after transfection with miR-18a mimics or antagomirs. RT-qPCR was used to measure the expression level of miR-18a, which was then normalized
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to RNU6B. Error bars indicate standard errors. p-value indicates the significance of difference by Student’s test. *P<0.05; **P<0.01. Figure 1D. Cell viavility assay of HCECs transfected with miR-18a mimics or antagomirs.
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MTT method was used to determine the cell viability of HCECs after transfection. x-axis represents time point post treatment or transfection, and y-axis represents average OD values of three biological replicates. Figure 2A. Expression of CTGF mRNAs in HCECs transfected with miR-18a mimics and antagomirs. Cells were transfected with miR-18a mimics, miR-18a antagomir, or sramble sequences. The group of cells transfected with scramble sequences has a parallel treated with 0.6 mg/mL SH to compare the effect of SH, miR-18a mimics and miR-18a antagomir on CTGF level. mRNAs were quantified and normalized to GAPDH, which was presented as fold change (y-axis). *P<0.05; **P<0.01. Figure 2B. Expression of CTGF proteins after transfection with miR-18a mimics and antagomirs. The group of cells transfected with scramble sequences has a parallel treated with 0.6 mg/mL SH to compare the effect of SH, miR-18a mimics and miR-18a antagomir on
ACCEPTED MANUSCRIPT CTGF level.. Western blot was conducted 2 days after transfection. Figure 2C. Sequence of CTGF mRNA 3’UTR and a mutant CTGF 3’UTR sequence for miR-18a.
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Figure 2D. Relative luciferase activity of miR-18a mimic+WT-CTGF-3’UTR vs. miR-18a
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mimic+MUT-CTGF-3’UTR. *P<0.01, vs Scramble
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Figure 3A, 3B. Knock-down efficiency of CTGF siRNA. mRNAs were quantified and normalized to GAPDH, which was presented as fold change (y-axis). Error bar: standard error; **: p<0.01.
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Figure 3C. Cell viability of CTGF knock-down HCECs.
x-axis represents time point post
treatment or transfection, and y-axis represents average OD values of three biological
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replicates, indicating cell numbers. Significance was test by Student’s test. *P<0.05; **P<0.01. Figure 3D. HCECs transfected with CTGF siRNA under microscopy. Cells were visualized
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under × 400 magnification.
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Figure 3E. Effect of CTGF knock-down on wound healing of HCECs. Images represent wouund healing assay from untreated HCECs and those transfected with CTGF siRNAs at 0
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and 24 hrs. Photographs were captured under ×40 magnification. Figure 3F. Quantification of migration distance of HCECs transfected with CTGF siRNA in
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wound healing assay. **P<0.01.
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ACCEPTED MANUSCRIPT Table 1. Overrepresented pathways of top 200 predicted target genes of miR-18a Observed
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Arf6 downstream pathway VEGF and VEGFR signaling
1575
network Plasma
1556
membrane
estrogen
signaling
ErbB1 downstream signaling
1571
mTOR signaling pathway
1619
Endothelins Beta1
integrin
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1602
cell
surface
cell
surface
interactions Integrin
family
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interactions
Integrin-linked kinase signaling
1488
CDC42 signaling events E-cadherin
signaling
in
the
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nascent adherens junction
1494
N-cadherin signaling events Regulation of nuclear SMAD2/3
1611
signaling
Cell junction organization
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936 935
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1546
1544
3.5E-07
5.73
Cell-Cell communication
3.5E-07
5.71
3.5E-07
22
5.69
3.5E-07
22
5.67
3.5E-07
22
5.67
3.5E-07
22
5.67
3.5E-07
22
5.67
3.5E-07
22
5.76
3.5E-07
22
5.95
5.6E-07
22
6.07
7.66E-07
13
2.89
2.63E-05
13
3.34
0.0001
7
1.21
0.0006
6
1.11
0.0023
6
1.34
0.0055
3
0.03
0.0074
3
0.52
0.0245
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focal adhesion kinase
1499
5.74
GMCSF-mediated
Signaling events mediated by
1517
22
22
Arf6 trafficking events
15744
3.5E-07
Nectin adhesion pathway events
1615
5.67
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1461
adjP
22
22
receptor signaling
1472
Expected
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Pathway
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ID
“adjP” represent Benjamini-Hochberg corrected p-value; “Observed” denotes the number of overlapped genes between queried gene set and pathway gene set, and “Expected” stands for the expected number of randomly sampled genes of same size with queried set in this pathway.
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Abbreviations miRNA: microRNA siRNA: small interference RNA CTGF: connective tissue growth factor SH: sodium hyaluronate HCECs: human corneal epithelial cells KGM: keratinocyte growth medium PBS: phosphate-buffered saline Arf6: ADP-ribosylation factor 6 CDC42: Cell Division Cycle 42 FRS2: Fibroblast growth factor substrate 2 COLA1A: Collagen, Type XXI, Alpha 1 VEGF: Vascular Endothelial Growth Factor A EGFR: Epidermal Growth Factor Receptor Rac1: Ras-Related C3 Botulinum Toxin Substrate 1 CD44: CD44 Molecule
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Highlights · miR-18a and miR-92 are significantly downregulated in sodium hyaluronate treated human corneal epithelial cells. · miR-18a was identified as effector in the stimulatory effect of sodium hyaluronate on HCECs proliferation and migration. · Functional enrichment of predicted target of miR-18a identified multiple pathways implicated in cell proliferation and migration. · CTGF was identified as the functional target of miR-18a
S0378-1119(16)30534-0 doi: 10.1016/j.gene.2016.07.008 GENE 41441
To appear in:
Gene
Received date: Revised date: Accepted date:
10 January 2016 12 May 2016 3 July 2016
Please cite this article as: Guo, Yingzhuo, Lu, Xiaohe, Wang, Hua, Downregulation of miR-18a induces CTGF and promotes proliferation and migration of sodium hyaluronate treated human corneal epithelial cells, Gene (2016), doi: 10.1016/j.gene.2016.07.008
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ACCEPTED MANUSCRIPT Downregulation of miR-18a induces CTGF and promotes proliferation and migration of sodium hyaluronate treated human corneal epithelial
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cells
a
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Yingzhuo Guoa,b, Xiaohe Lua*, Hua Wangb
Department of Ophthalmology, Zhujiang Hospital, Southern Medical University, Guangzhou
510280 , Guangdong Province, China
Department of Ophthalmology& Optometry, Hunan Provincial People's Hospital, Changsha,
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b
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410005, Hunan Province,China.
*Corresponding author: Xiaohe Lu, Department of Ophthalmology, Zhujiang Hospital,
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Southern Medical University, No. 253 Industrial Road，Guangzhou 510280 , Guangdong
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Province, China (Email: [email protected] )
ACCEPTED MANUSCRIPT ABSTRACT
Properly controlled corneal epithelial wound healing is critical for health of
cornea, which involves cell proliferation, migration, anchoring and differentiation. Sodium
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hyaluronate (SH) has been proven to exert beneficial pharmacological effect on corneal wound
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healing, though the underlying mechanism remained open to investigation. MicroRNAs (miRNAs) are small single-stranded RNAs that could bind to 3’UTR of mRNAs of target genes. The multi-target regulation of miRNAs may favor treatment of corneal wound given the
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complicated processes implicated in the healing process, which has inspired initiatives to
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develop miRNA therapy in corneal wound healing. In this light, we used miRNAs profiling to detect whether miRNAs are also implicated in the mechanism underlying the stimulatory effect
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of SH on corneal epithelial wound healing. We found miR-18a was most susceptible to SH
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treatment, the target prediction of which were enriched in a bunch of pathways implicated in
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corneal wound healing. Connective tissue growth factor (CTGF) was found to be overrepresented in most significant enriched pathways and was experimentally confirmed as a
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bona fide target of miR-18a, which modulated cell migration and proliferation of human corneal epithelial cells.
Keywords: corneal epithelial wound healing; sodium hyaluronate; miR-18a; CTGF
ACCEPTED MANUSCRIPT INTROCUTION Corneal wound healing encompasses a sequential and highly coordinated events, including cell death, proliferation, migration, differentiation and extracellular matrix remodeling[1]. The
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corneal epithelium is the outermost part of the ocular surface, where the epithelial cells are
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constantly regenerating to maintain a clear and proper functioning cornea. Corneal epithelial
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wounds can be caused by mechanical abrasion, chemical burn or ophthalmic surgeries, which, if not adequately healed, can result in corneal haze, ulcer, perforations, refractory epithelial defect or blindness[2]. Therefore, rapid and proper regeneration of epithelium is of vital
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importance for corneal wound healing. Epithelial healing at the corneal surface is a dynamic process that involves proliferation of basal epithelial cells, centripetal and circumferential
underneath connective tissue[3].
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migration of epithelial cell sheets, and anchoring of newly generated epithelium to the
Sodium hyaluronate (SH) is a naturally occurring polysaccharide of high viscoelasticity, which
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has been used in ophthalmic surgery to protect the corneal epithelium and endothelium[4]. It
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has also been incorporated into artificial tears to treat dry eye[5]. Studies of animal corneal
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wound model showed that SH could stimulate cell migration, adhesion, and proliferation during corneal epithelial wound healing[6], which was found to be peculiar for SH since other polysaccharides, such as chondroitin sulphate, keratan sulphate and heparan sulphate, could
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not produce similar effect. This indicates that the therapeutic effect of SH on corneal epithelial wound healing is not only dependent on its viscoelasticity, but also pharmacological factors[7], though the underlying mechanism remained controversial[3]. MicroRNAs are 22-24 nt non-coding single-stranded RNAs that can bind to sequences on 3’UTR of target mRNAs, which lead to perturbed translation or degradation of target mRNAs[8]–[10]. Over a decade of exploration, miRNAs are proved to be powerful gene expression regulators that mediate various biological processes[11], [12]. The importance of miRNAs in corneal wound healing has been highlighted by the evidence that some miRNAs promote healing but others inhibit it[9], [13]–[16]. Due to the miRNA-miRNA variability, they can usually target multiple genes[17]. Since wound healing is a complex process, the multi-target regulation of miRNAs may favor treatment that requires combinatorial solutions. Indeed, the application of anntagomir-based therapy has been performed in correcting the aberrance of
ACCEPTED MANUSCRIPT epithelial wound healing in human diabetic organ-culture corneas[13], [16]. Recently, Lilly et al. reported
hyaluronan treatment could promote production
of
miR-10b,
leading to
Rho-kinase-associated breast tumor cell invasion[18]. This prompted us to investigate
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changes of the miRNA profile in corneal epithelial cells (HCECs) in response to SH, in an attempt to address the mechanism underlying the accelerated wound healing process.
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In this study, we demonstrated subtle alteration in miRNAs profile in HCECs following SH treatment, and identified miR-18a as most susceptible to treatment, which was substantially downregulated. Subsequent target prediction combined with functional enrichment analysis
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identified a few pathways that are possiblely targeted by miR-18a, which offers candidates for further investigation. In “Arf6 downstream pathway” that ranked at the top of predictions, we
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verified CTGF as a functional target in wound healing, the knock-down of which was found to significantly inhibit proliferation and migration of HCECs.
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MATERIAL AND METHODS
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Cell culture and transfection with miR-18a antagomir, mimic and CTGF siRNA Human corneal epithelial cells (ThermoFisher) were maintained in human keratinocyte growth
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medium (KGM-2; Invitrogen, CA) and incubated at 37℃, humid air containing 5% CO2. Cells were then seeded onto 96-well plates and grown to about 80–90% confluence. The morphology of cultures were evaluated on daily basis by phase contrast microscopy. Three 2+
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days before experiment, cells were transferred to KGM-2 supplemented with 1mM Ca for 12h, and then washed with sterile phosphate-buffered saline (PBS). At day four, cell migration was evaluated and six similar cultures were chosen and randomly assigned to control or experimental group, each containing three biological replicates. Control group and experimental group were cultured in KGM-2 alone and KGM-2 with SH (0.6mg/ml), respectively. Transfection Scrambled control or miR-18a (or miR-92) antagomir/mimic (ThermoFisher, Ambion) were introduced into HCECs using Lipofectamine RNAi Max (ThermoFisher) for 6 h. Cells were harvested for assays two days after transfection. For CTGF siRNA transfection, lentivirus expressing CTGF siRNA was constructed and amplified as described by Wang et al.[19].
ACCEPTED MANUSCRIPT Pooled lentiviral siRNA and scrambled siRNA (Scr-siRNA) were prepared for targeting human CTGF and as negative control, respectively. MiRNA microarray analysis
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Agilent 2100 Bioanalyzer was used to assess RNA quality. Finally 100 ng of total RNA was processed for use on the microarray using the FlashTagTM HSR labeling kit (Genisphere LLC)
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as per manufacturer’s protocols, in which the cRNA was generated and biotinylated to hybridized to the GeneChip miRNA Array (Affymetrix). Affymetrix Model 3000 scanner wasused to scan to array accordingly. MiRNA QC tool software (Affymetrix) was used to
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generaate expression value which was normalized and log transformed. Differential expression was detected by R package limma, which was defined as log2 fold change>=1.5 or
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<=-1.5 (adjP<0.05).
Target Analysis and pathway common analysis
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TargetScan Release 7.0 (http://www.targetscan.org/) was employed to predict targets of
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miR-18a, which is based on searching for the presence of 8mer, 7mer, and 6mer sites that match the seed region of each miRNA. The 2,898 predictions were ranked by “cumulative
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weighted context ++ score”, and top 200 were selected for pathway common analysis to identify functional clusters that are of biological significance related to miR-18a and may contain target genes. Webgestalt[20], an online webserver for functional enrichment analysis,
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was adopted to perform pathway common enrichment using hypergeometric test. Pathway analysis was initially used to identify significant pathways linkage to differentially expressed genes. Likewise, as miR-18a may target multiple genes, the overrepresented pathway for prediction may address biological implication of miR-18a, within which the target will be included. The minimum genes contained in each pathway was set to “2” and significance “p=0.05” corrected by Benjamini-Hochberg (FDR) method. Quantitative RT-PCR TRIzol Reagent (Invitrogen) was used to extract total RNA from HCECs, the quality of which was confirmed. Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA from 10 ng total RNA. The expression of miRNAs or mRNA (CTGF) was quantified by RT-PCR using 7500 Fast Real-Time PCR System (Applied Biosystems), which was then normalized to U6B small nuclear RNA (RNU6B) or GAPDH depending on the
ACCEPTED MANUSCRIPT experiment. Cell viability assay The number of viable cells was determined by Methylthiazolyldiphenyl-tetrazolium bromide
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assay (Sigma-Aldrich). Cells were washed with PBS to remove non-adherent cells, and plated
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at 5,000 per cell, which was incubated for 6 hours. MTT reagent was added to the well, 10μL
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per well. Incubate the wells for 3 hrs at 37℃ until purple precipitate was visible. The cells were then added with 100μL detergent reagent per well and placed in the dark for 2 hrs, after which the absorbance at 570nm were taken.
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Western blot analysis
Cells were lysed in NP-40 buffer containing 150mM NaCl, 1.0% NP-40, 50mM Tris-HCl and
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protease inhibitors (Roche) and 1mM phenylmethyl sulfonylfluoride for 30 minutes on ice. The supernatant was subject to protein denaturation with buffer containing 2% sodium
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dodecylsulfate (SDS; Sigma), was subsequently analyzed using SDS-PAGE and monoclona
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antibody against CTGF. The resultant gel was then washed and covered by appropriate horseradish peroxidase-conjugated Ig secondary antibodies. Enhanced chemiluminescence
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(GE Healthcare) was used to detect the staining. Luciferase activity assay
CTGF 3’UTR and its mutated counterpart sequence plasmids were constructed. Cells were 5
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seeded in a 24-well plate at cell density of 1×10 cells/well. After 24 hours of culture, cells were cotransfected with firefly luciferase reporter plasmids respectively containing wild-type or mutant CTGF 3’UTR pRL-TK vector expressing Renilla luciferase, and miR-18a or miRNA negative control via Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured after 36 hours by Dual Luciferase Reporter Assay (Promega, US). Each tranfection was performed twice in triplicate.
Scratch wound healing assay Twenty-four hours after transfection with scrambled control antagomir/mimic (100 nmol/L), miR-18a antagomir/mimic, or CTGF siRNA, and a 200μL pipette tip was used to make a straight scratch on the culture, followed by washing with Hanks medium until no floating cells were present. The scratched cultures were then maintained in serum free medium.
ACCEPTED MANUSCRIPT Photographs were taken immediately and at 24h after scratches were made. Statistical analysis Results are expressed as mean values±std., and a Student’s t-test was used for testing
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statistical significance. P<0.05 is considered as significant, whereas P<0.001 represents extreme significance.
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RESULTS
Sodium hyaluronate (SH) modulates expression of microRNA in corneal epithelial cells
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Microarray was conducted to profile the microRNA expression in human corneal epithelial cells (HCECs) after 2 days exposure to 0.6mg/ml SH[21]. Differentially expressed microRNAs were There was only a few
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defined as those with log2 fold change>=1.5 or <=-1.5 (adjP<0.05).
significant variation in treated cells compared with control (Figure 1A). In total, 16 miRNAs were deregulated, among which miR-18a and miR-92 were most highly downregulated, and
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miR-210, miR-26b, and miR-378 showed the largest fold change. The number of induced
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miRNAs and their repressed counterparts were similar, however, the repressed miRNAs showed higher absolute fold change, implying that one or more particular biological processes
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may be activated, to mediate control of corneal wound healing. We then performed RT-PCR to confirm the differentially expressed miRNAs. As anticipated, miR-18a was dramatically repressed (logFC (log-transformed fold change) = -1.987983, p=2.05E-13; Figure 1B), next to
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which was miR-92 (logFC=-1.228615, p=6.52E-12; Figure 1B).
Antagomir and mimics of miR-18a respectively enhanced and deterred the proliferation of HCECs Since miR-18a showed predominantly large absolute fold change among all deregulated miRNAs, we reasoned that miR-18a may be essential for promoting wound healing in SH treated cells. To examine the critical role of miR-18a in corneal wound healing, we used mimics and antagomir to transfect HCECs to see if the cell proliferation will be regulated accordingly. The transfection efficiency was confirmed by quantifying miR-18a for every treatment or transfection (Figure 1C). The cell viability was measured for 3 days to monitor the cell growth. The HCECs transfected with miR-18a mimics showed cell growth rate slower than
ACCEPTED MANUSCRIPT HCECs transfected with scramble sequences,
while those transfected with miR-18a
antagomir displayed accelerated growth rate. (Figure 1D). Putative targets of miR-18a were enriched in Arf6 downstream pathway
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Since miRNAs function by mediating partial or entire degradation of mRNAs of target genes, determination of target genes are critical for elucidating the role of miR-18a in HCECs. To
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address this, we adopted Targetscan, a tool based on searching for the presence of 8mer, 7mer, and 6mer sites that match the seed region of each miRNA, to predict target genes of miR-18a. There were 2, 898 transcripts predicted as target of miR-18a, which were
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ranked by “cumulative weighted context ++ score”, a measure indicating the confidence of prediction (data not shown).
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We then adopted enrichment analysis to identify functional clusters containing genes that mediate re-epithelialization of HCECs. To narrow down the gene set, top 200 genes were
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selected for pathway common analysis ( the list is available at : http://www.targetscan.org)[20].
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These predicted genes were enriched in multiple pathways implicated in cell proliferation, cell cycle, and cell mobility (Table 1). These pathways include “Arf6 downstream pathway”, “VEGF
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and VEGFR signaling network”, “Nectin adhesion pathway”, “Arf6 trafficking events”, “Endothelins”, and “E-cadherin signaling in the nascent adherens junctionn”. In particular, we noticed that “Arf6 downstream pathway“ and “Arf6 trafficking events” are both involved in the
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formation of lamellipodia at the leading edge of migrating corneal epithelial cells and in the growing cones of corneal nerve fibers[22]. This agreement validates the roles of these pathways in corneal wound healing. “Arf6 downstream pathway” contained 22 putative target genes, including components of cell division cycle, such as CDC42 (cell division cycle 42) and CCND2 (cyclin D2), and of cell growth, such as FRS2 (fibroblast growth factor receptor substrate 2) and CTGF (connective tissue growth factor). CTGF is a target of miR-18a CDC42 and CCND2 are critical component of cell cycle and cell proliferation, and are ubiquitous in various cell types[11], [23], [24]. We presented that miR-18a mediated both cell proliferation, therefore, we reasoned that CDC42 and CCND2 may not be the major factors that control corneal wound healing after treatment of SH, since migration entails not only cell proliferation but also cell motility, such as cell-to-cell communication. Both FRS2 and CTGF
ACCEPTED MANUSCRIPT were reported to be associated with cell migration[25]–[28]. Recently, Blalock et al. revealed that expression of CTGF could mediate effect of TGF-beta induction of COLA1A synthesis in corneal fibroblasts, which promotes corneal wound healing[29]. In this light, we postulated that
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miR-18a might modulate biological processes by suppressing CTGF. To testify our speculation, we performed both quantitative RT-PCR and western blotting to detect CTGF expression in
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HCECs after being transfected with miR-18a mimics and antagomir, respectively. Transfection with miR-18a antagomir resulted in a remarkable increase in CTGF protein, while the HCECs transfected with miR-18a mimics present substantial reduction (Figure 2A, B). In addition, the
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level of CTGF in SH treated cells was dramatically elevated compared to untreated cells (scrambled control), suggesting that CTGF is likely a bona fide target of miR-18a and may
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respond to SH treatment.
In addition, we further conducted dual luciferase reporter assay to verify whether CTGF is a
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bona fide target of miR-18a. The CTGF mRNA 3’ UTR or CTGF 3’UTR with mutation
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sequence plasmids (Figure 2C) were cotransfected with miR-18a or negative control into HCECs. After 36 hours, the luciferase activity was measured. The result showed that HCEC
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cells transfected with miR-18a mimics had luciferase activity significantly lower than control, suggesting that CTGF is a target of miR-18a (Figure 2D). Silencing of CTGF counteracted the migration and proliferation enhanced by SH
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Although we demonstrated that CTGF is the target of miR-18a in HCECs, as miRNAs usually target multiple genes, the role of CTGF in SH treated HCECs remained to be determined. We measured the changes in cell viability and scratch wound healing rate of CTGF silenced HCECs treated with SH. Knockdown efficiency was confirmed at mRNA and protein level (Figure 3A, 3B). The control demonstrated significantly higher growth rate during observation, while the CTGF silenced cells display slow growth during the first two days of observation (Figure 3C, 3D). As anticipated, in wound healing, CTGF ablated HCECs showed impaired cell mobility (Figure 3E, 3F), which is corresponding to protein level of CTGF (Figure 3A). Impairment of proliferation and migration resulting from CTGF deficiency indicates the critical role of CTGF in the underlying mechanism. DISCUSSION
ACCEPTED MANUSCRIPT The regulatory role of miRNAs had been well documented in the studies of eye development and pathology. Mutations of dicer, a pivotal enzyme responsible for cleaving double-stranded RNA into double stranded pre-miRNA, could result in defects in normal development of retina,
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lens, and cornea[30]–[32]. In addition to global aberrance in regulation of miRNA due to deficient dicer, specific miRNAs have also been identified as functional in corneal epithelial
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development, which indicates their therapeutic potentials. For example, miR-145 modulates the corneal epithelium formation and maintenance of epithelial integrity via targeting ITGB8[33]. miR-31, which is preferentially expressed in epithelial, downregulates factor
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inhibiting hypoxia-inducible factor-1 (FIH-1) and thus affects glycogen metabolism and keratinocyte differentiation in corneal epithelial cells[34], [35]. These discoveries highlight the
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importance of miRNAs in corneal epithelial homeostasis. Indeed, the miRNAs expression display a tissue specific pattern in cornea. miR-184 is highly expressed in epithelium of mouse
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lens and central cornea[36]; miR-124 and miR-204 were concordantly expressed in all lens
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epithelial cells, while miR-205 was expressed all over the corneal epithelium[37]. In addition, the short length of miRNAs preclude its one-on-one targeting to respective target genes, which
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means that a miRNA could target multiple genes and vice versa. Therefore, a holistic scanning of miRNAs would facilitate identification of effector miRNAs. The nuance difference between miRNA profiles of SH treated cells and control attested to this necessity, since the few
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differentially expressed miRNAs might be missed without complete profiling. Corneal epithelial healing involves highly orchestrated events including cell proliferation, migration, adhesion and differentiation in order to maintain the integrity and health of corneal epithelial surface[38]. The application of SH in treating corneal wound healing is well-established. Hyaluronic acid and high potassium ion concentration artificial tears could promote corneal epithelial wound healing in scraping model[39], and the stimulatory effect of HA on migration of rabbit corneal epithelial cells in vitro has been documented[40]. Recently, miRNA was highlighted as one of the novel technologies developed to manipulate corneal wound healing. In this light, we performed microarray to detect deregulation of miRNAs in response to SH treatment in HCECs, in an attempt to uncover the potential role of miRNAs in physiological mechanism underlying the enhancement of wound healing by SH. The concentration of 0.6mg/ml was used in the present study in keeping with previous studies,
ACCEPTED MANUSCRIPT which has been through gradient testing of optimal concentrations between 0.4~1.0mg/ml[3]. The largely consistent expression level of the majority of miRNAs between SH treated cells and control implied the effect and safety of using SH in treating corneal epithelial injury[3],
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since it’s likely that the proliferation and migration were promoted at the expense of less perturbation in other biological pathways.
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Among the virtually maintained miRNAs, miR-18a and miR-92 exhibited prominent downregulation, which was then verified by RT-qPCR where miR-18a showed predominantly lower fold change. This contrast projects the major regulatory role of miR-18a under SH
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treatment, and the inhibitory function of miR-18a was evidenced by the observation of deterred
transfected with miR-18a antagomir.
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proliferation in cells transfected with miR-18a mimics and enhanced proliferation in cells
Target gene prediction of miR-18a by TargetScan renders over two thousand hits, from which
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we selected top 200 predictions of context++ model. This model was more predictive than any
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published model and even equivalent with in vivo crosslinking approaches in miRNA target prediction[41]. Pathwaycommon enrichment was employed to infer functional clusters that
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may contain target genes. We arrived at six pathways highly implicated in cell migration and proliferation, including “Arf6 downstream pathway”, “VEGF and VEGFR signaling network”, “Nectin adhesion pathway”, “Arf6 trafficking events”, “Endothelins”, and “E-cadherin signaling
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in the nascent adherens junctionn”, among which “Arf6 downstream pathway” ranked at the top. Vascular endothelial growth factor (VEGF), was originally identified as a growth factor in angiogenesis. However, it has also been implicated in mediating cell proliferation in various epidermal tumors. Autocrine VEGF signaling synergizing with EGFR in tumor cells has the ability to promote epithelial cancer development[42]. Recently, several studies demonstrated the stimulatory effect of VEGF on multiple biological processes of the wound healing cascade, including vascularization, epithelization and collagen deposition[43]. Therefore, it is reasonable to postulate a potential similar role of VEGF in corneal epithelium. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, was found in human corneal epithelium, and more interestingly, in rabbit tear fluid[44]. Hitoshi et al. highlighted the therapeutic value of ET-1 in remedy of corneal epithelial defects by demonstrating elevated wound healing rate in culture corneal epithelial cells treated with ET-1 containing eyedrops[45]. Cell proliferation is critical for
ACCEPTED MANUSCRIPT continuous renewal of cornea and recovery from corneal trauma and keratoplasty, while during corneal epithelial wound healing, the progenitor corneal epithelial cells at the periphery may undergo formation of lamellipodia, protrusions that favor exploration of local environment and
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cognate cells, followed by epithelial to mesenchymal transition (EMT), a hallmark feature in the process of tumor invasion and wound healing[46], [47]. Both nectin and cadherin-based
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cell-cell adhesions are play important roles in adherens junctions of epithelial cells[48]. Whether the nascent junction between cells will grow and mature or decompose to promote proliferation is determined by Rac1 activity[49]. Transient presence of Rac1 is essential for
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membrane exploration at nasceent junctions, but thereafter, it would deter the maturation and undermine the junction. Felipe et al. revealed that during epithelial cell scattering, a process
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characteristic of EMT, cell-cell dissociation could be induced by initial decrease of Rac1, and this downregulation is ARF6-dependent[50]. Although common pathway enrichment was
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utilized to pin-point functional modules that may contain target genes of miR-18a, considering
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the large average number of miRNA targets, we cannot rule out that a subset of predictions may be bona fide targets. In addition, the engagment of these overrepresented pathways in
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the regulatory netwok of wound healing reinforces this possibility. Arf6 signaling was deemed to be most pertinent to epithelialization among the enriched pathways. Of the 22 genes contained in “Arf6 downstream signaling”, we chose CTGF as
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candidate, which was verified experimentally. Indeed, a previous study reported that miR-18a has a functional site in the CTGF 3’-UTR, and its etopic expression dramatically diminished CTGF mRNA levels as well as proteins[51], which was correlated with survival in glioblastoma. This agreement suggests that corneal wound healing and cancer metastasis share common biological processes. CTGF was identified as an important component in corneal wound healing and scar formation[52]. The role of CTGF in corneal wound healing was further evidenced by the nullification of CTGF deficiency on cell mobility proved to be stimulated by SH, which is consistent with a recent study by Daniel and colleagues where they observed immediate upregulation of CTGF in the epithelium at the wound margin and maintained high expression of CTGF during re-epithelialization[53]. They also reported a 40% reduction in the corneal re-epithelialization rate in conditional CTGF knock-out mouse model. Similarly, Lee et al. found that CTGF expression was higher in synovial cells deprived of serum but treated with
ACCEPTED MANUSCRIPT higher concentration of hyaluronic acid[54]. Although the molecular processes elicited by increase of CTGF which were conducive to epithelialization remained to be delineated, our study uncover a mechanism whereby SH demonstrated its therapeutic value in corneal
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epithelial wound healing, which implicates reduction of miR-18a and increase of CTGF therefrom. In addition, the observation that HA could expidite the synthesis of ECM and the cell
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could bind with CD44, the endogeneous receptor of HA, to facilitate cell migration[54], offers an alternative explanation for the stimulatory effect of SA on corneal epithelial wound healing. Recently, Yuchen et al. found that downregulation of CD44 inhibited transcription of YAP
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downstream effectors CTGF, Cyr61 and EDN1[55]. In the present study, the miR-18a mimics cannot completely block the expression of CTGF, indicating another mechanism controling
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expression of CTGF, which may involve CD44. Taken together, our study provides a complementary explanation for the stimulatory effect of SH on corneal epithelial wound
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healing.
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In summary, we observed subtle alterations in miRNA profile in HCECs following SH treatment, and miR-18a stood out as most susceptible. Target prediction yields an enormous amount of
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candidates, which was narrowed down by functional enrichment analysis. The six overrepresented pathways are highly implicated in multiple components of corneal epithelial wound healing, such as cell proliferation, formation of lamellipodia, and EMT, suggesting that
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multiple genes in those pathways may be bona fide targets of miR-18a, though we finally fixed on CTGF which was verified experimentally. Although the underlying mechanism remained to be delineated, our study revealed its role in the therapeutic effect of SH on corneal epithelial wound healing in relation to reduction of miR-18a. In addition, the functional clusters identified based on predicted targets of miR-18a may offer candidates for futher exploration. CONFLICT OF INTEREST None.
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LEGENDS Figure 1A. Differentially expressed miRNAs between SH treated cells and control.
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Volcano plot of miRNA profile. Red dots represent miRNAs are were detected as significantly
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expressed by p-value, and green dots represent non-siginificantly expressed miRNAs. y-axis
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denotes negative log10 transformed p-values, and x-axis present log2 transformed fold change. The differential expressed miRNAs in our study were defined as those with significant log2 fold change>=1.5 or <=-1.5.
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Figure 1B. Quantitative RT-PCR verification of miR-18a and miR-92 against control. miR-18a and miR-92 were most dramatically downregulated. y-axis indicates fold change
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against control, and x-axis denotes the expression level of miR-18a or miR-92 in SH treated cells. Error bar indicates standard errors. p-value indicates the significance of difference by
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Student’s test.
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Figure 1C. Expression of miR-18a after transfection with miR-18a mimics or antagomirs. RT-qPCR was used to measure the expression level of miR-18a, which was then normalized
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to RNU6B. Error bars indicate standard errors. p-value indicates the significance of difference by Student’s test. *P<0.05; **P<0.01. Figure 1D. Cell viavility assay of HCECs transfected with miR-18a mimics or antagomirs.
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MTT method was used to determine the cell viability of HCECs after transfection. x-axis represents time point post treatment or transfection, and y-axis represents average OD values of three biological replicates. Figure 2A. Expression of CTGF mRNAs in HCECs transfected with miR-18a mimics and antagomirs. Cells were transfected with miR-18a mimics, miR-18a antagomir, or sramble sequences. The group of cells transfected with scramble sequences has a parallel treated with 0.6 mg/mL SH to compare the effect of SH, miR-18a mimics and miR-18a antagomir on CTGF level. mRNAs were quantified and normalized to GAPDH, which was presented as fold change (y-axis). *P<0.05; **P<0.01. Figure 2B. Expression of CTGF proteins after transfection with miR-18a mimics and antagomirs. The group of cells transfected with scramble sequences has a parallel treated with 0.6 mg/mL SH to compare the effect of SH, miR-18a mimics and miR-18a antagomir on
ACCEPTED MANUSCRIPT CTGF level.. Western blot was conducted 2 days after transfection. Figure 2C. Sequence of CTGF mRNA 3’UTR and a mutant CTGF 3’UTR sequence for miR-18a.
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Figure 2D. Relative luciferase activity of miR-18a mimic+WT-CTGF-3’UTR vs. miR-18a
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mimic+MUT-CTGF-3’UTR. *P<0.01, vs Scramble
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Figure 3A, 3B. Knock-down efficiency of CTGF siRNA. mRNAs were quantified and normalized to GAPDH, which was presented as fold change (y-axis). Error bar: standard error; **: p<0.01.
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Figure 3C. Cell viability of CTGF knock-down HCECs.
x-axis represents time point post
treatment or transfection, and y-axis represents average OD values of three biological
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replicates, indicating cell numbers. Significance was test by Student’s test. *P<0.05; **P<0.01. Figure 3D. HCECs transfected with CTGF siRNA under microscopy. Cells were visualized
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Figure 3E. Effect of CTGF knock-down on wound healing of HCECs. Images represent wouund healing assay from untreated HCECs and those transfected with CTGF siRNAs at 0
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and 24 hrs. Photographs were captured under ×40 magnification. Figure 3F. Quantification of migration distance of HCECs transfected with CTGF siRNA in
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wound healing assay. **P<0.01.
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ACCEPTED MANUSCRIPT Table 1. Overrepresented pathways of top 200 predicted target genes of miR-18a Observed
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Arf6 downstream pathway VEGF and VEGFR signaling
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network Plasma
1556
membrane
estrogen
signaling
ErbB1 downstream signaling
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mTOR signaling pathway
1619
Endothelins Beta1
integrin
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cell
surface
cell
surface
interactions Integrin
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Integrin-linked kinase signaling
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CDC42 signaling events E-cadherin
signaling
in
the
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nascent adherens junction
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N-cadherin signaling events Regulation of nuclear SMAD2/3
1611
signaling
Cell junction organization
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3.5E-07
5.73
Cell-Cell communication
3.5E-07
5.71
3.5E-07
22
5.69
3.5E-07
22
5.67
3.5E-07
22
5.67
3.5E-07
22
5.67
3.5E-07
22
5.67
3.5E-07
22
5.76
3.5E-07
22
5.95
5.6E-07
22
6.07
7.66E-07
13
2.89
2.63E-05
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3.34
0.0001
7
1.21
0.0006
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1.11
0.0023
6
1.34
0.0055
3
0.03
0.0074
3
0.52
0.0245
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focal adhesion kinase
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5.74
GMCSF-mediated
Signaling events mediated by
1517
22
22
Arf6 trafficking events
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3.5E-07
Nectin adhesion pathway events
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receptor signaling
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Abbreviations miRNA: microRNA siRNA: small interference RNA CTGF: connective tissue growth factor SH: sodium hyaluronate HCECs: human corneal epithelial cells KGM: keratinocyte growth medium PBS: phosphate-buffered saline Arf6: ADP-ribosylation factor 6 CDC42: Cell Division Cycle 42 FRS2: Fibroblast growth factor substrate 2 COLA1A: Collagen, Type XXI, Alpha 1 VEGF: Vascular Endothelial Growth Factor A EGFR: Epidermal Growth Factor Receptor Rac1: Ras-Related C3 Botulinum Toxin Substrate 1 CD44: CD44 Molecule
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Highlights · miR-18a and miR-92 are significantly downregulated in sodium hyaluronate treated human corneal epithelial cells. · miR-18a was identified as effector in the stimulatory effect of sodium hyaluronate on HCECs proliferation and migration. · Functional enrichment of predicted target of miR-18a identified multiple pathways implicated in cell proliferation and migration. · CTGF was identified as the functional target of miR-18a