Activation of LncRNA FOXD2‐AS1 by H3K27 acetylation regulates VEGF‐A expression by sponging miR‐205‐5p in recurrent pterygium

Abstract LncRNA FOXD2‐AS1 is abnormally expressed in many diseases. However, the molecular mechanisms whereby FOXD2‐AS1 is involved in recurrent pterygium remain unknown. Here, qRT‐PCR was performed to quantify FOXD2‐AS1 expression, while CCK‐8, flow cytometer and neoplasm xenograft assays were used to investigate its function. Dual‐luciferase reporter, RIP and RNA pull‐down assays were conducted to address the relationship between FOXD2‐AS1, miR‐205‐5p and VEGF‐A, while ChIP assays were used to detect H3K27 acetylation at the FOXD2‐AS1 promoter. FOXD2‐AS1 expression was up‐regulated in recurrent pterygium tissues. Moreover, a high FOXD2‐AS1 expression was associated with advanced stages, increased microvessel density and shorter recurrent‐free survival. In addition, ROC analysis showed that FOXD2‐AS1 is a valid predictor of recurrent pterygium. Furthermore, we show that FOXD2‐AS1 induced proliferation and inhibited apoptosis in a cell line derived from recurrent pterygia (HPF‐R) at least partially through the regulation of the miR‐205‐VEGF pathway. In addition, the up‐regulation of FOXD2‐AS1 was attributed to the H3K27 acetylation at the promoter region. In conclusion, FOXD2‐AS1 is activated via its H3K27 acetylation and regulates VEGF‐A expression by sponging miR‐205‐5p in recurrent pterygium. Our results may provide a basis for the development of new therapeutic targets and biomarkers for recurrent pterygium.

novel biomarkers for recurrence have constituted the major goals in pterygium research.
Long noncoding RNAs (lncRNAs) are defined as non-protein-coding RNAs longer than 200 nucleotides. Several studies have associated lncRNAs to biological processes in human cancers, such as cell proliferation, apoptosis, invasion and angiogenesis. [3][4][5] In recent years, studies on the role of lncRNAs in pterygium have also made some progress. Liu et al identified 3066 up-regulated and 1646 down-regulated lncRNAs in pterygium tissues compared with paired adjacent normal conjunctival tissues. 6 Lan et al reported that linc-9432 is up-regulated in pterygium and regulates the levels of differentiation-related transcripts in fibroblasts. 7 In addition, lncRNAs have also been described as candidate biomarkers for prognosis. For instance, lncRNA-MEG3 is a proven biomarker for retinoblastoma and cervical cancer. 8,9 Therefore, noncoding RNAs are a new promising source of disease biomarkers that can be applied in clinical practice.
Overexpression of FOXD2-AS1 causes malignant cell proliferation and the down-regulation of several tumour suppressor genes. 10,11 However, the expression level and function of FOXD2-AS1 in recurrent pterygium have not been reported yet. In addition, it remains unclear whether FOXD2-AS1 levels can be used as a biomarker for recurrent pterygium. Thus, we sought to answer these questions.
In this study, we found overexpression of FOXD2-AS1 in recurrent pterygium tissues and that the expression levels were associated with disease stage, microvessel density (MVD) and recurrent-free survival. Moreover, FOXD2-AS1 had predictive value in recurrent pterygium. In addition, function assays revealed that FOXD2-AS1 induces cell proliferation and inhibits cell apoptosis both in vitro and vivo. Furthermore, our results suggest that FOXD2-AS1 may act as a competing endogenous RNA (ceRNA) to regulate the miR-205-VEGF pathway in HPF-R cells and that H3K27 acetylation activates FOXD2-AS1 expression. In conclusion, FOXD2-AS1 promotes pterygium growth, at least partially, through the regulation of the miR-205-VEGF pathway. Thus, we propose FOXD2-AS1 as a potential biomarker for recurrent pterygium. information is summarized in Table 1.

| Cell lines established from of recurrent pterygia (HPF-R)
Freshly collected recurrent pterygium tissues were cut into small sections, washed in Hanks solution, and incubated in DMEM medium (Invitrogen, Carlsbad, California, US) with 100 mmol/L sorbitol (Sigma, Missouri, US) and 50 μg/mL dispase II (Invitrogen) for 60 minutes at 37°C. Then, pterygium cells were dissociated using TryPLE reagent (Invitrogen) for 5 minutes at 37°C. The isolated cells were cultured in DMEM medium supplemented with 10% FBS and gentamicin (50 g/mL) at 37°C in a humidified atmosphere with 5% Purification of cell: First, we used flow cytometry to separate p63 (+) and pan cytokeratin (+) pterygium cells. [12][13][14] Next, we observe the cell morphology under an inverted phase-contrast microscope (Olympus, Tokyo, Japan) and scrape off other cells using cell scraper. Stable cells passaged for 3 to 7 generations were used in the experiments. C646 (Selleck Chemicals, Houston, TX, USA) was used at 10 μmol/L for 48 hours as required.

| Establishment of stable cell lines
Lentiviruses carrying FOXD2-AS1 plasmids or expressing shRNA against FOXD2-AS1 were constructed by Genechem (Shanghai, China). Transfection of HPF-R cells with lentiviral vectors was performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Puromycin (2 μg/mL)-resistant clones were picked and separated, and green fluorescent protein signals were checked using fluorescence-activated cell sorting. The shRNA sequences used are listed in Additional file 1: Table S1.

| Immunofluorescent staining
Cells were washed with cold-PBS three times, fixed with 4% formaldehyde for 60 minutes and permeabilized using 0.1% triton- x for 30 minutes. After blocking with 10% normal goat serum for 1 hour at room temperature, the samples were incubated with pri- Technology) before image acquisition using a fluorescence microscope (Olympus, Tokyo, Japan).

| Cell apoptosis assay
Cell apoptosis was detected using the Annexin V-

| Neoplasm xenograft assay
Twelve BALB/c nude mice (6-8 weeks old) were randomly and equally divided into four groups (Three mice per group).

| Dual-luciferase reporter assay
Cells were cultured in 12-well plates and co-transfected with luciferase reporter constructs of target genes (wild type or mutated type 3'-UTRs respectively, Genearray Biotechnology, China) and miR-205-5p mimics using Lipofectamine 3000 (Invitrogen). After 48 hours, luciferase activity was measured using the Dual-Luciferase reporter assay system according to the manufacturer's instructions (Promega, Fitchburg, WI, USA). Relative luciferase activity was normalized to Renilla luciferase activity. Biotin-labelled miR-205-5p-WT, miR-205-5p-MUT and control probe were purchased from Geneseed Biotech (Shanghai, China). Cells were lysed with lysis buffer and incubated with specific probes. Then, the cell lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen, San Diego, CA, USA) to pull-down the biotin-labelled RNA complex according to the manufacturer's protocol. The bound RNAs were purified using Trizol for qRT-PCR analysis.

| Chromatin immunoprecipitation assay (ChIP)
ChIP was conducted using EZ ChIP™ Chromatin Immunoprecipitation

| Statistical analysis
SPSS software package 13.0 was used to perform statistical analyses, and the results are shown as the mean ± SD from three separate experiments. Data were analysed using two-tailed Student's t test and χ 2 test, Fisher's exact test or Wilcoxon test, as appropriate.
Pearson correlation analysis was performed to investigate correlations. A receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value for recurrence prediction.
The odds ratio (OR) was calculated using logistic regression analysis, and Hazard Ratio (HR) was calculated using Cox regression analysis. Recurrent-free survival rates were calculated using the Kaplan-Meier method with the log-rank test applied for comparison. The statistical significance level was set at P < 0.05. All experiments were conducted in triplicates.

| Correlation between FOXD2-AS1 and clinicopathologic characteristics of pterygium patients
qRT-PCR analysis demonstrated that the expression of FOXD2-AS1 was significantly higher in pterygium tissues than in adjacent conjunctiva tissues (P < 0.01, Figure 1A). Further, we found that patients at advanced stages tended to have higher expression of FOXD2-AS1 (P < 0.05, Figure 1B) and increased microvessel density (P < 0.05, Figure 1C). FOXD2-AS1 was not related to either the age or the gender of pterygium patients. The odds ratio (OR) of FOXD2-AS1 for MVD and advanced stage is shown in Figure 1F.

| Clinical value of FOXD2-AS1 in predicting pterygium recurrence
First, the expression of FOXD2-AS1 was significantly up-regulated in recurrent pterygium patients compared to primary pterygium patients (P < 0.05, Figure 1A). Thus, we decided to assess whether FOXD2-AS1 could be used as a predictive tool for recurrent pterygium, for which we used receiver operating characteristic and specificity of 77.9% (P = 0.003, Figure 1D).
To evaluate the prognostic value of FOXD2-AS1, recurrent pterygium patients were separated into 'high-risk' (n = 13) and 'low-risk' (n = 9) groups according to the best cut-off level of FOXD2-AS1. Kaplan-Meier survival curves showed that patients in the high-risk group had shorter recurrent-free survival (P = 0.0014, Figure 1E). In addition, Cox regression analyses revealed a Hazard Ratio (HR) for recurrent FOXD2-AS1 of 1.857 Figure 1F). Collectively, our results indicated that the FOXD2-AS1 signature is a potential biomarker for the prediction of pterygium recurrence.

| FOXD2-AS1 suppresses apoptosis and promotes neoplasm growth in HPF-R cells
Flow cytometry analysis indicated that the early apoptosis rate of HPF-R1 cells, which overexpress FOXD2-AS1, was significantly decreased compared to that of control cells. In contrast, the early apoptosis rate of HPF-R2 cells was significantly increased after FOXD2-AS1 knockdown (P < 0.05, Figure 3A). Furthermore, the FOXD2-AS1-overexpressing xenografts progressed much faster and grew much larger than the controls in vivo. Contrarily, FOXD2-AS1knockdown xenografts grew much smaller and slower than the controls in vivo (P < 0.05, Figure 3B).  Figure 4C).

| FOXD2-AS1 targets miR-205-5p directly
First, we used bioinformatics tools to identify the target miRNAs of FOXD2-AS1 and discovered a complementary sequence between FOXD2-AS1 and miR-205-5p ( Figure 5A). Next, we confirmed that miR-205-5p expression was significantly down-regulated in HPF-R1 cells and significantly increased in HPF-R2 cells compared with their control groups, respectively (P < 0.05, Figure 5B). We also used pterygium tissues and adjacent conjunctiva tissues to perform qRT-PCR assays, which revealed that miR-205-5p was not only lower in primary pterygium tissues than in adjacent conjunctiva tissues, but also lower in recurrent pterygium tissues than in primary pterygium tissues (P < 0.05, Figure 5C). Moreover, Pearson's correlation analysis showed that the levels of miR-205-5p and FOXD2-AS1 were negatively correlated (R = −0.701, P < 0.001, Figure 5D).

Additionally, RIP experiments showed enrichment of miR-205-5p
and FOXD2-AS1 in immunoprecipitated Ago2, which is a key protein with ceRNA function, compared with the control IgG (P < 0.05, Figure 5G).

| VEGF-A is a direct target of miR-205-5p
First, we predicted that VEGF-A could be a downstream target of miR-205-5p using bioinformatics tools ( Figure 6A). Next, we examined the effects of miR-205-5p mimics and inhibitor. RT-PCR results showed that the expression of miR-205-5p in the miR-205-5p mimics group and miR-205-5p inhibitor group was 3.8-fold and 0.54-fold that of their control groups, respectively (P < 0.05, Figure 6B).  Figure 6C). In contrast, the mRNA and protein levels of VEGF-A were dramatically up-regulated in HPF-R cells in which miR-205-5p was inhibited, compared with the control cells (P < 0.05, Figure 6D). Indeed, luciferase reporter assays with WT and MUT type VEGF-A 3'-UTR binding sites for miR-205-5p also showed decreased luciferase activity with WT VEGF-A 3'-UTR upon miR-205-5p overexpression, while MUT VEGF-A 3'-UTR binding sites had no effect on luciferase activity (P < 0.05, Figure 6E).

| H3K27 acetylation activates FOXD2-AS1 expression
To further understand the mechanism behind the high FOXD2-AS1 levels in HPF-R cells, we performed a bioinformatics analysis (http:// genome.ucsc.edu/) and found a high enrichment of H3K27ac in the FOXD2-AS1 promoter ( Figure 8A). Further, we conducted a ChIP assay using 22 paired tissues and found a significantly increased level of H3K27ac at the FOXD2-AS1 promoter in recurrent pterygium tissues compared with adjacent conjunctiva tissues (P < 0.05, Figure 8B). Furthermore, the H3K27ac enrichment was also significantly increased in HPF-R cells compared with HconEpiC cells (conjunctival cell line) (P < 0.05, Figure 8C). As expected, FOXD2-AS1 expression levels were significantly up-regulated in HPF-R cells compared with HconEpiC cells (P < 0.05, Figure 8D). Moreover, the enrichment in H3K27ac was dramatically lower in C646-treated HPF-R cells compared with the control cells (P < 0.05, Figure 8E,F).

| D ISCUSS I ON
Aberrant expression of FOXD2-AS1 is involved in cancer initiation, progression and metastasis. 10,11 Pterygium cells are tumour-like transformed cells that compared to normal fibroblasts, grow much more rapidly in medium without high concentrations of serum and can grow in a semisolid agar. 15 However, the role of FOXD2-AS1 in pterygium progression and prognosis has not been clarified. In this study, we observed up-regulation of FOXD2-AS1 expression in pterygium tissues. Further, we found that the FOXD2-AS1 level was positively correlated with advanced stages and increased MVD in pterygium tissues. Logistic regression analysis demonstrated that FOXD2-AS1 is a risk factor for advanced stages and increased MVD.
Indeed, an inflamed stage and increased MVD are crucial factors for recurrent pterygium. Further, we found that FOXD2-AS1, which is related to the inflamed stage and increased MVD, is associated with poor recurrent-free survival. Collectively, our data demonstrate that FOXD2-AS1 may be involved in pterygium progression and recurrence.
F I G U R E 4 FOXD2-AS1 regulates the expression of VEGF-A. A, B, Immunofluorescence staining and Western blot analyses of FOXD2-AS1 effect on the expression of VEGF-A and β-catenin. C, Correlation between FOXD2-AS1 and MVD using Pearson's correlation assay in pterygium tissues (n = 126). Scale bar, 50 µm. *P < 0.05 Growing evidence suggests that lncRNAs such as FOXD2-AS1 could serve as potential biomarkers with high sensitivity and specificity for disease detection and diagnosis. 9,10,16 Based on this, we explored the potential application of FOXD2-AS1 as a biomarker for the prediction of recurrent pterygium. First, we found that FOXD2-AS1 expression levels were significantly higher in recurrent pterygium tissues than primary pterygium tissues. Next, Kaplan-Meier analysis confirmed that patients with high FOXD2-AS1 expression had significantly shorter recurrent-free survival than those with low expression. Cox regression analyses also proved that FOXD2-AS1 was a risk factor for recurrence. Last, the specificity of FOXD2-AS1 in predicting pterygium recurrence was confirmed in the ROC curve.
Thus, we suggest that FOXD2-AS1 may be an ideal biomarker for recurrent pterygium. The pathogenesis of pterygium is an active process associated with cellular proliferation and angiogenesis. The evidence indicates that VEGF is increased in pterygium and may contribute to its progression and recurrence by increasing angiogenesis and growth.
For instance, Wu 19 reported that VEGF can activate fibroblasts in pterygium by overexpressing low-density lipoprotein receptors.  Second, we revealed the regulatory effect of miR-205-5p on VEGF-A. We found that miR-205-5p reduces both mRNA and protein VEGF-A levels in HPF-R cells. In addition, the results from the luciferase reporter assay also revealed that VEGF-A may be one of the direct targets of miR-205-5p in HPF-R cells. Last, we explored the mechanism behind the high FOXD2-AS1 expression in recurrent pterygium. Recent studies have shown that the aberrant expression of lncRNAs is attributed to acetylation-mediated transcriptional activation. [27][28][29] Histone acetylation leads to the weakening of the DNA-histone interaction and the subsequent activation of transcription. 30 Therefore, we analysed the promoter region of FOXD2-AS1 by bioinformatics analysis and identified that H3K27ac was highly enriched in this region. Furthermore, we confirmed that the enrichment level of H3K27ac at the FOXD2-AS1 promoter was notably increased in both HPF-R cells and recurrent pterygium tissues, resulting in FOXD2-AS1 up-regulation. The histone acetylation process is controlled by histone acetyltransferases and histone deacetylases. Therefore, we investigated whether the H3K27ac modification is involved in FOXD2-AS1 expression using C646 (histone acetyltransferase inhibitor). As expected, C646 reduced the H3K27ac enrichment at the FOXD2-AS1 promoter, and consequently, resulted in a decreased expression of FOXD2-AS1.
Overall, our results strongly support that the enrichment of H3K27ac at the FOXD2-AS1 promoter region leads to the up-regulation of FOXD2-AS1 expression.
In summary, our study demonstrates for the first time that FOXD2-AS1 is activated by H3K27 acetylation and that this activation leads to enhanced proliferation and suppression of apoptosis in cell lines established from recurrent pterygium fibroblasts.
Moreover, we show that this action is exerted through the regulation of the miR-205-VEGF pathway. Based on our results, we propose FOXD2-AS1 as a potential novel therapeutic target and diagnosis biomarker for recurrent pterygium.

ACK N OWLED G EM ENT
The

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest were disclosed.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.