Distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data
© Tong et al; licensee BioMed Central Ltd. 2009
Received: 04 August 2008
Accepted: 10 March 2009
Published: 10 March 2009
Pterygium is a common ocular surface disease characterized by fibrovascular invasion of the cornea and is sight-threatening due to astigmatism, tear film disturbance, or occlusion of the visual axis. However, the mechanisms for formation and post-surgical recurrence of pterygium are not understood, and a valid animal model does not exist. Here, we investigated the possible mechanisms of pterygium pathogenesis and recurrence.
First we performed a genome wide expression analysis (human Affymetrix Genechip, >22000 genes) with principal component analysis and clustering techniques, and validated expression of key molecules with PCR. The controls for this study were the un-involved conjunctival tissue of the same eye obtained during the surgical resection of the lesions. Interesting molecules were further investigated with immunohistochemistry, Western blots, and comparison with tear proteins from pterygium patients.
Principal component analysis in pterygium indicated a signature of matrix-related structural proteins, including fibronectin-1 (both splice-forms), collagen-1A2, keratin-12 and small proline rich protein-1. Immunofluorescence showed strong expression of keratin-6A in all layers, especially the superficial layers, of pterygium epithelium, but absent in the control, with up-regulation and nuclear accumulation of the cell adhesion molecule CD24 in the pterygium epithelium. Western blot shows increased protein expression of beta-microseminoprotein, a protein up-regulated in human cutaneous squamous cell carcinoma. Gene products of 22 up-regulated genes in pterygium have also been found by us in human tears using nano-electrospray-liquid chromatography/mass spectrometry after pterygium surgery. Recurrent disease was associated with up-regulation of sialophorin, a negative regulator of cell adhesion, and never in mitosis a-5, known to be involved in cell motility.
Aberrant wound healing is therefore a key process in this disease, and strategies in wound remodeling may be appropriate in halting pterygium or its recurrence. For patients demonstrating a profile of 'recurrence', it may be necessary to manage as a poorer prognostic case and perhaps, more adjunctive treatment after resection of the primary lesion.
Global gene expression has been used successfully to elicit biological behavior in different soft tissue tumors. Pterygium as a human disease, noted to be more prevalent than 20% of some populations,[2, 3] is of immense biological interest for a few reasons.
First, the pathogenesis of this condition is hotly debated. Hypothesis driven approaches have not resolved the relative importance of competing mechanisms for this disease. Theories that have been proposed include inflammatory influence, degeneration of connective tissue, genetic instability, angiogenesis, redox-related toxicity, cellular proliferation, aberration of apoptosis, exuberant wound healing, altered lipid metabolism, mast cell infiltration. and stem cell dysfunction. Conventional approaches to disease mechanism, by virtue of their narrow focus, were not helpful to assess relative contribution of widely heterogenous processes. Furthermore, a fundamental issue about the diseased tissue remains un-resolved in this context: the origin of the epithelium overlying pterygial lesions, though suspected to be conjunctival in origin, is not entirely certain.
Second, the spectrum of tumor size and behavior is tremendous, ranging from the inconspicuous lesion barely encroaching on the peripheral cornea, to the rapidly growing, menacing tumors that obscure the visual axes and threaten vision. In some contexts, additional molecular events. may drive the original tumor to behave differently from the original lesion. It is also intriguing that unlike frank malignancies, these lesions, however fast growing, do not erode through the full thickness cornea, highlighting the presence of distinct processes from those manifested in malignant tumors.
Third, pterygium is a mixed soft tissue tumor that is strongly associated with non-ionising ultraviolet radiation. Unlike the case of cutaneous melanomas, the cell type that responded to the environmental trigger may not be epithelial but rather, the fibro-vascular component – a truly unusual phenomenon in human tumor biology because ultraviolet radiation-induced tumors encountered in humans are epithelial in origin and ultraviolet radiation effects on keloids, if any, are generally inhibitory.
Lastly, the inability of researchers to discover an analogous tumor in animals or reconstitute the disease in organ cultures imply that pterygium involves specie-specific mechanisms mediated by in-vivo cell-cell or cell-matrix interactions. The molecules involved in this disease may have therefore arisen from divergent evolution in the human ocular surface.
In view of the controversies on the multiple mechanisms of pterygia formation, we advocate an unbiased, global gene expression approach to decipher the dominant gene expression patterns that may underlie specific molecular events of interest. Recently, the genome-wide microarray data in whole tissue pterygium as well as microarray data limited to cultured pterygial fibroblasts, have been published. We have performed a study using gene microarray on recurrent pterygium, primary pterygium and un-involved conjunctiva, examined the gene subsets which were differentially regulated, compared our findings with these previous microarray studies,[16, 17] and discussed some of the findings in the light of a complementary proteomics approach.
In clinical practice, the treatment of this condition is surgical excision. However, some cases aggressively recur after surgery. Conjunctival auto-grafting as an adjunctive procedure may reduce recurrence, though the explanation for this is not entirely clear. For these reasons, we speculate that a non-hypothesis driven approach may also be useful to discover gene expression signatures that predispose lesions to a more aggressive phenotype. Such information will benefit clinicians, who can appropriately anticipate otherwise 'un-expected' biological behavior in their treatment of this disorder.
Antibodies against MUC5AC, keratin 6 and CD24 were from Labvision, Neomarkers, Fremont, CA, USA, anti-MSMB antibodies were from US Biologicals, Massachusetts, MA, USA, anti-CEACAM5 and anti-NR4A2 antibodies were from Abcam, Cambridge, UK.
Samples used for the Study
The procurement and use of both human tissues in this study was in compliance with the tenets of the Declaration of Helsinki. The study was approved by the Institutional Review Board of Singapore Eye Research Institute. Written informed consent was obtained from donors after explanation of the nature and possible consequences of the study. Human tissues samples were obtained from patients diagnosed with primary pterygium and came from different races, Chinese, Malay and Indian. All patients underwent pterygium excision in conjunction with the use of an upper bulbar conjunctival free autograft placed over the site of the original lesion. All pterygia specimens used were nasal pterygia. The whole pterygium tissue and a small portion of the conjunctival patch (approx. 1 × 3 mm) from the superotemporal conjunctiva were collected and were rapidly frozen in liquid nitrogen after removal and stored at -150°C. For immunohistology purpose, the samples were collected on ice and embedded in Optimal Cutting Temperature (OCT, Sakura, USA) in the laboratory.
The procedure for the microarray experiment has previously been published. All microarray chips and related protocols and equipment for the processing of these chips were from Affymetrix Inc., Santa Clara, CA. The human genome GeneChip U133A consisting of more than 22000 probe sets was used for this study.
A group of 8 primary pterygium samples, harvested from 4 males (aged 40 to 50 years old) and 4 females (aged 50 to 60 years old) was used in this experiment. Another group of 4 control conjunctiva tissues, each pooled from 4 individual samples, was used as controls to the diseased tissues. Considering that only a tiny piece of the conjunctiva tissue could be obtained from each patient during the surgery, pooling of 4 conjunctiva tissues was necessary to obtain enough starting material.
Total RNA was extracted using TRIzol Reagent (Invitrogen, CA) and purified with RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Five micrograms of each purified RNA sample were prepared according to the Affymetrix standard protocol. Fifteen micrograms of biotin-labelled cRNA using BioArray RNA Transcript Labelling Kit (ENZO Life Sciences, NY) were fragmented and the appropriate volume injected separately into the probe array chips.
The transcripts were hybridized onto the immobilized oligonucleotide sequence on array for 16 hours at 45°C under 60 rpm rotation using GeneChip Hybridization Oven 640. Washing and Strepavidin-staining steps were performed using Affymetrix Fluidics Station 450. The chips were scanned using GeneChip Scanner3000 and the image data were further analyzed using Microarray Suite v.5.0. Data analysis included pre-processing and normalization before identification of differentially expressed genes. Pre-processing adjusted for non-specific binding and background noise, whereas normalization removed systematic variation in the data due to effects other than biological differences. We used the Robust Multi-array Average (RMA) model  to extract the gene expression signals from probe intensities without taking the mismatch probe signals into consideration. Cross-array normalization was performed using the intensity-based log ratio median method  with the first array as the reference. Gene-level normalization was performed by normalizing all samples to the median of the expression level of the control (un-involved conjunctiva) samples. The data were annotated using gene annotation headings, bioprocesses and molecular functions from the NetsAffx database http://www.affymetrix.com/analysis/index.affx.
Analysis of Microarray Data
The data reported in this study have been deposited in NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/ with GEO series accession number GSE2513). Data were visualised and explored using the GeneSpring GX 7.3 platform (Agilent Technology, Redwood City, CA).
For selection of differentially expressed genes, the modified t-statistic (SAM)  with 100% of the standard deviation percentile as the fudge constant was used. The threshold for significantly changed genes was set at a false discovery rate (FDR) of 5%. After delineating a list of differentially expressed genes, we performed various types of analysis to search for a pattern of global gene expression. The methods ranged from K-means clustering, to the construction of hierarchical dendrograms on a subset of genes, as well as the visualisation of gene expression pattern summarised by principal component analysis. In additional to analysis of differentially expressed genes, we employed the Gene Set Enrichment Analysis (GSEA) method for identification of those pathways that were more affected in pterygium tissues. The goal of GSEA is to determine whether members of a gene set tend to occur toward the top or bottom of the list, in which case the gene set is correlated with the phenotypic class distinction. In contrast to the methods for extraction of differentially expressed genes, GSEA considers the collective up- or down-regulation of a gene set rather than individual genes. The GSEA software package downloaded from the Board Institute's website was used for the identification of activated or deactivated pathways in primary pterygial and conjunctival tissues. Microarray data for 8 primary pterygial samples and 4 samples of conjunctival tissue were used. Various datasets from publicly accessible databases (Biocarta, STKE, PubMed and KEGG) were dissected into over 300 gene sets. The two phenotypic classes used were pterygium and uninvolved conjunctiva tissues.
As an independent method of analyzing signaling pathways in pterygium, we exported the list of genes significantly up-regulated or down-regulated by 2 fold into the Pathway Studio 5.0 software (Ariadne Genomics Inc, Rockville, MD). The option 'Find all shortest paths between selected entities' was used on the up-regulated and down-regulated genes sequentially. The number of connectivities was limited to 2 and the genes mapped into known pathways displayed on a chart, and the relevant relationships listed in a table. The purpose of performing this analysis was to identify important upstream regulators of the differentially expressed genes as well as downstream effectors, in an unbiased fashion, based on known biological knowledge.
Methods used to extract and analyse tear proteins from patients who had undergone surgery for pterygium have already been described. Wherever possible, the fold change data for genes corresponding to detected tear proteins were tabulated.
Real Time Reverse Transcription Polymerase Chain Reaction
Six pairs of independent pterygium and uninvolved conjunctiva tissues were used in the real time reverse transcription polymerase chain reaction (qPCR) experiment. One microgram of the each total RNA preparation was reverse-transcribed to single stranded cDNAs using an oligo-dT primer with Superscript II Rnase H Reverse Transcriptase (Invitrogen, USA). Primer pairs specific to each gene used in the qPCR are listed in Additional file 1. qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The thermal cycling conditions were as follows: 95°C for 10 min, 45 cycles at 95°C for 30 s and 60°C for 1 min. Data obtained from qPCR were analyzed using the comparative CT method as previously described by Livak. Paired t-statistics with p-value < 0.05 was used to determine whether the qPCR results in pterygial tissue were significantly different from those in un-involved conjunctiva.
A separate group of paired pterygial and conjunctival tissue samples from three patients were used for immunohistochemistry. Sections were cut at 5 μm thicknesses from blocks of freshly frozen pterygium and matched un-involved conjunctiva embedded in the OCT. The post-fixed sections were incubated with the primary antibody at 4°C overnight. Specific antibodies against the following were used: MUC5AC, MSMB, CD24, CEACAM5, keratin 6 and NR4A2. After 3 washes with PBS, secondary antibody used was either fluorescein isothiocyanate (FITC)- or Rhodamine- conjugated anti IgG (Santa Cruz, USA), incubated with the sections for 40 minutes at room temperature. After 3 final washes each section was mounted with in a fluorescence mounting medium (DAKO, Denmark). Images were captured using a 40× Achrostigmat lens on an Axioplan2 microscope equipped with an AxioCam MR camera (Carl Zeiss, Germany).
Global Gene Expression Profiling and analysis of pterygium versus conjunctiva
Gene expression analysis
Genes significantly changed by at least 2-fold difference in pterygium (p < 0.05)
Up-regulated in pterygium
Down-regulated in pterygium
Gene Ontology Analysis
Some immune response and transcription factors were down-regulated in primary pterygium. Examples of the former include immunoglobulin heavy chain genes, whereas examples of the latter include ATF3, BTG2, ERG1, FOS, FOSB, JUN, NR4A1 and NR4A2. Other down-regulated genes include those encoding for transport proteins, ie., HBB and HBD, and those involved in stress-response, including DUSP1 and GADD45B.
Proportion of significantly changed genes by functional category.
Gene Ontology (GO)
Extracellular region (5576)
Extracellular matrix (31012)
Intermediate filament cytoskeleton (45111)
Intermediate filament (5882)
Muscle development (7517)
Transporter activity (5215)
Structural molecule activity (5198)
Extracellular matrix structural constituents (5210)
Oxidoreductase activity acting on paired donors (16705)
Analysis of enriched pathways
The only significantly enriched pathway in un-involved conjunctiva was related to apoptosis. The genes in this pathway include the calpains (CAPN1, CAPN2, CAPN3, CAPNS1) and bcl-antagonist of cell death (BAD2).
Pathway analysis to identify upstream and downstream signals
K-means cluster analysis supplemented with GO browser results.
No of Genes
Status in pterygium
Major function by GO
Response to stimulus
Cell adhesion, response to wounding
Differences between primary and recurrent pterygium
Distinguishing recurrent from primary pterygia (p < 0.05), list of genes down-regulated or up-regulated by at least 2-fold.
A. Name of down-regulated genes
No of folds down-regulated
B. Name of up-regulated genes
No of folds up-regulated
Validation of gene microarray data
Comparison of microarray data with tear proteins
Gene microarray data corresponding to detected tear proteins.
Name of gene corresponding to tear protein
No of fold change
A. Genes significantly regulated in pterygium
Prolactin-induced protein I
S100A8 (Calgranulin A)
Defensin alpha A1
Hepatocellular carcinoma associated protein (PIGR)
Clusterin (Apolipoprotein J)
Vitamin D binding protein
Orosomucoid 1 (alpha 1 acid glycoprotein)
Proline rich Peptide
Lipophilin C (SCGB2A1)
B. Genes not significantly regulated in pterygium
Immunoglobulin kappa light
Proline rich 4 (lacrimal)
Nasopharyngeal carcinoma associated proline rich protein 4
Immunoglobulin J chain
Complement factor H
Secretary leukocyte protease inhibitor
Proline rich protein 5
Coagulation factor H (thrombin)
Basic proline rich protein
A global gene expression analysis of pterygium showed distinct differences between primary pterygium and uninvolved conjunctiva. Several pathways were significantly affected in pterygium. These were: increase in the production of extracellular matrix, structural proteins, mitotic proteins, and protein involved in tissue invasion.
Recurrent pterygia demonstrated a different signature composed of other perturbed genes. For example, the COL4A6 (AL031177) and the RAB6B (BC002510) were down- and up-regulated respectively in recurrent pterygia compared to primary pterygia. The former encodes for one of the 6 subunits of collagen IV, a major component of the basement membrane, whereas the latter is a RAS family oncogene. The finding suggests that recurrence is a distinct biological phenomenon from the formation of primary pterygium, even though in general, we did not detect obvious microscopic changes between primary and recurrent pterygia.
Microarray analysis of gene expression is a useful approach for understanding the molecular mechanism of disease. We used both qPCR and immunohistochemistry to show that both RNA level and protein levels of specific mediators were dysregulated, validating the results from the gene microarray approach. Some 22 genes corresponding to tear proteins detected in pterygium subjects were significantly up or down-regulated in pterygium relative to conjunctival tissue. Many of the processes discovered in this study are highly novel and were not previously associated with pterygium, for example, immunohistochemistry show that cell adhesion molecules (ie., CD24) may be increased but abnormally localized in the nuclei in pterygium epithelium and therefore cell adhesion properties may be disturbed. Another example is the evidence for Goblet cell dysfunction. In pterygium, there was elevation of transcript and protein expression of MUC5AC and MSMB. MUC5AC is a well-known marker of conjunctival mucous-secreting Goblet cells, and MSMB is a cutaneous squamous cell carcinoma related protein that is associated with respiratory tract Goblet cells.
Comparison with previous studies
Comparison of microarray analysis between two studies
Fold change in John-Aryankalayil et al
Fold change in our study
Significantly regulated in current study (p < 0.05)
SILV (silver homolog)
Von Willebrand factor
S100 A9 (calgranulin B)
mRNA, clone AL050154
Prostaglandin F2alpha receptor
Not significantly regulated in current study (p > 0.05)
Carbonic anhydrase CA1
Casein kinase II
Myosin heavy chain II
Previous studies[28, 29] have shown that gene expression profiles in tumor and wound response were similar, lending support to our opinion that wound response is the major theme in global pterygial gene expression.
Possible mechanisms of pterygia formation
We believe that our data support a predominantly wound healing pattern of gene expression in pterygium. Genes encoding for extracellular matrix, structural and adhesion molecules including wound healing related proteins, collagen subtypes, keratin 6A and fibronectin were significantly up-regulated in pterygium. Other up-regulated proteins like small proline rich protein 1B (SPRR1B), CD24, S100 calcium binding protein, SPARC, TFF1, SPRR1B and SERPINB13 also govern the wound healing process and cornification of epithelium, again, supporting the hypothesis of aberrant wound response in pterygium. Fibronectin alternatively splice transcript with EDA, up-regulated in our pterygial specimens, was shown to be up-regulated in wound healing. In order to further validate this finding, semi-quantitative PCR was performed on paired pterygial and conjunctival tissue specimens from 3 patients to examine the level of fibronectin transcript with EDA. The results show that pterygium expressed higher levels of fibronectin transcript with EDA compared to uninvolved conjunctiva (Figure 8C).
Our data also suggest that in pterygia formation, there may be increased cellular proliferation and motility on one hand, and reduced cell death on the other. Genes up-regulated in pterygium include fibronectin (FN1), CEACAM5 (CEA), CD24, SPARC, MSMB and TFF1. CEACAM5 (CEA), a common cancer marker, was up-regulated 3.8-fold in pterygium. SPARC was known to have anti-adhesive property, and induced cancer cell motility. The transcripts coding for microseminoprotein (MSMB) or PSP94 was up-regulated 3.6-fold, whereas transcripts for calcium binding protein S100A8 was up-regulated 5.7-fold in pterygium. Interestingly, in our analysis of tears from patients with pterygium, S100 proteins were also detected. Trefoil factors promote restitution of epithelial cells and are abundantly secreted onto the mucosal surface rapidly after mucosal injury. In contrast to the above, genes coding for apoptosis (TGM2, IGFBP3 and DUSP1) were down-regulated in pterygium, reinforcing the over-proliferative tendency in pterygium.
The stress-inducible transcription regulator genes including ATF3, BTG2, EGR1, ERG2, FOS, JUN, NR4A1 and NR4A2 were surprisingly, down-regulated in pterygium relative to uninvolved conjunctiva. One has to bear in mind however, that our tissue specimens may represent chronic UV stimulation. The expression of these transcription factors may very well be elevated shortly after the commencement of the initial stimulus.
The transcript level of immunoglobulin subunit in pterygium was relatively lower as compared to uninvolved conjunctiva. This does not support a role for B cell mediated immunity in pterygium formation. However, the co-expression of some immunoglobulin genes (Figure 4) may have some biological significance, such as an immunological basis for the inappropriate wounding response.
Possible mechanisms of pterygia recurrence
One hypothesis is that a low level of inflammation may stimulate epithelial and fibroblast cell migration due to factors such as SPN [see Additional file 3A]. This allows continuation of inflammatory factors and cytokines to be produced, potentiating a cycle [see Additional file 3B]. Factors such as SPN are unique to recurrence only, since they were up-regulated compared to primary pterygium cases.
In an alternative hypothesis, the formation of an exuberant scar or a 'go' signal, may or may not be coupled with a reduction of 'stop' or inhibitory signals. One such 'stop signal' may be the MSMB protein. Any up-regulation of this anti-metastatic gene in pterygium could give an inhibiting signal to slow the growth rate in pterygium. When pterygium is surgically removed, this inhibiting factor may also be reduced, encouraging the remnants of fibrous tissue to proliferate more aggressively, attaining the phenotype of recurrent pterygium. This may also explain the success of conjunctival autographs as adjunctive procedure after excision of the lesion in the prevention of recurrence. The graft tissue may 'replenish' any surgically-induced loss of 'stop' signals. In support of this hypothesis, the level of MSMB was significantly depressed in recurrent compared to primary pterygia.
Strengths and Limitations
The strengths of this study include the use of a variety of analytical approaches to interpret the microarray data. The tissue specimens have been harvested in a center which manages a high volume of such disease. Samples have been processed in a very standardized fashion, obtained from patients with accurate clinical diagnoses.
One limitation of the study is that we did not evaluate p53 and related genes such as the Vasoendothelial growth factor (VEGF). Pterygia specimens contained a mixture of cell types whereas un-involved conjunctiva consisted largely of epithelial tissue. Therefore, any observations concerning differential gene expression could be related to differences in composition of cells as well as to pathological processes.
We did not notice regional differences in the intensity of CD24 immunofluorescent staining along the length of the pterygium epithelium on longitudinal section [see Additional file 7]. However, we cannot conclusively state that there is no difference in the pattern of CD24 expression between the head and the body of the pterygium. Since the limits of the pterygium body was defined clinically by the surgeon, we were uncertain if the bulk of the pterygium body was available for immunohistology.
Since we did not obtain temporally located pterygia in this study, our findings may not be applicable to this less frequently occurring type of pterygia.
Specimens analysed were at the later stage of pterygium formation when surgical removal was needed. No data at the early stage of disease were available for investigation.
In addition to these limitations, there are inherent limitations with hieuristic and even more deterministic methods of analyzing global gene expression. For example, the results of the k-means clustering depend arbitrarily on the initial number of clusters and the number of iterations. However, we attempt to reduce the effects of these shortcomings by employing a variety of different data-mining techniques and interpreting results as a whole and not in isolation. We drew conclusions based on findings that are consistent between different procedures and refrained from over-interpretation of isolated anomalies.
If changes in global gene expression in cases of recurrent pterygium do not occur until a very late time point, it may not be possible to predict recurrence of the condition by analysing the tissue obtained during the time of first excision. However, the gene expression data may still be useful for understanding the biology of recurrence. The tear protein analysis also has limitations. It is difficult to determine whether the detected proteins were physiological, related to the surgical trauma, or arising from the disease. Nevertheless the 22 significantly up-regulated genes that have products in human tears may be potential biomarkers for the disease.
This study illustrates that an unbiased global gene expression approach is useful to address the disease mechanisms in a controversial condition. Further studies on more specimens may allow the use of a sub-set of genes to prognosticate lesions. Non-surgical treatment for pterygium currently does not exist. Since wound healing and matrix dysregulation is a major theme, known modulators of wound healing may be explored in pterygium in a targeted way, using topical application. Other novel processes require further evaluation. Studies in the regulation of cell adhesion by CD24 and mucous processing pathways are required to understand pterygium formation. Studies are also required to elicit the origin of the overlying epithelium in pterygium tissue.
Nevertheless, our study suggests that after resection of the initial tumor, clinicians can modify treatment in selected patients based on objective criteria after analysing tumor gene expression or tumor proteins.
Based on differential gene profiling in pterygium, it can be concluded that an aberrant wound healing process is the major pathogenetic process in pterygial formation. The other secondary processes such as cornification and attempted barrier formation may be compensatory in nature. This contrasts sharply with frank malignant neoplasms, where gene expression signatures are dominanted by increased proliferation, reduced apoptosis, cell cycling anomalies and genomic instability. The existence of an aggressive phenotype to account for post excision recurrence may be related to an imbalance of growth signals rather than due to mere prolongation of the stimulus that initiated primary pterygial formation.
(complementary) deoxyribonucleic acid
false discovery rate
Gene expression omnibus
Gene set enrichment analysis
optimal cutting temperature
- (q) PCR:
(quantitative) polymerase chain reaction
Robust Multi-array Average
(complementary) ribonucleic acid.
We would like to thank Tracy Chai for performing the microarray and immunofluorescent staining experiments and Dr Wong Yong Wee for contributing to the analysis in the paper. The study is funded by Singapore Biomedical Research Council (BMRC) 03/1/35/19/231 and National Medical Research Council (NMRC) IBG. BMRC has approved the submission of this manuscript for publication.
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