MiR-199a-3p/5p participate in TGF-β and EGF induced EMT in pterygium by targeting DUSP5/MAP3K11 CURRENT STATUS: UNDER REVIEW

Background: Recently, it has been reported that miRNA is correlated with pterygium, however its exact mechanism in pterygium is unrevealed and require further investigation. Methods: The differential expression of miRNA in pterygium was profiled using microarray and validated with quantitative real-time PCR (qRT-PCR). Human conjunctival epithelial cells (HCEs) were cultured and treated with TGF-b and EGF. Western blot and immunohistochemistry were carried out to detect epithelial-mesenchymal transition (EMT) markers. Wound healing and transwell assay were used to determine cell migration ability, while apoptosis was determined by flow cytometry. The target genes of miR-199a were confirmed by the dual-luciferase reporter assay. Results: TGF-b and EGF induced EMT in HCEs to mimic the pathogenesis of pterygium. MiR-199a-3p and miR-199a-5p induced EMT in HCEs, whose respectively downstream targets DUSP5 and MAP3K11 hindered EMT in EMT-HCEs in turn. TGF-b and EGF induced EMT promotion and target genes suppression, could be promoted by miR-199a-3p and miR-199a-5p, while impeded by miR-199a-3p and miR-199a-5p inhibitors. The expression levels of miR-199a and target genes were further validated in pterygium tissues, which were consistent the results in cell model. Bioinformatics analysis indicated the miR-199a-3p/5p-DUSP5/MAP3K11 was belong to MAPK signalling pathway in pterygium. Conclusions: TGF-b and EGF probably induced EMT of HCEs through miR-199a-3p/5p-DUSP5/MAP3K11 axis, which explained the pathogenesis of EMT in pterygium and might provide new targets for pterygium prevention and therapy. and 29 control conjunctiva tissues. All pterygium samples were obtained from patients who diagnosed with primary pterygium undergoing a surgical resection and control conjunctiva tissues were collected from patients who underwent cataract surgery from December 2014 to October 2019 at Zhongnan Hospital of Wuhan University. Samples were stored at -80°C immediately after surgery until used. with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. Membranes were washed in TBS-T and exposured using the electrochemical luminescence (ECL) system. Protein loading was normalized by GAPDH.


Background
Pterygium is a common ocular surface disease with a triangular-shaped lesion growing in the limbal conjunctiva and progressive invasion of the cornea, affecting nearly 200 million people globally and the prevalence can even be as high as 22% in some countries [1][2][3][4]. Ultraviolet light exposure and ocular irritation were widely considered as the dominant risk factors of pterygium, and it caused the inactivation of p53. Inactivated p53 leaded to epithelial-mesenchymal transition (EMT), contributing to the pathogenesis of this disorder [5][6][7]. Nowadays, conjunctival auto-transplantation is the most common treatment for pterygium, but the recurrence rates range from 2-39% [8][9][10]. Recently, studies have linked aberrant miRNAs expression to EMT, but the precise contribution of miRNAs in the pathogenesis of pterygium has not been well documented [11][12][13].
The process of EMT was usually embodied as conjunctival epithelial cells lose the polarity and cell-cell adhesion, and obtain the migratory ability to become mesenchymal cells, which was verified as one of the most significant procedure of pterygium [21][22][23][24]. TGF-β and EGF were found to be two significant inducement in the process of EMT in various diseases [24][25][26][27]. Both TGF-β and EGF have been reported to be overexpressed in pterygium. TGF-β signalling stimulates fibroblasts migration, proliferation and myofibroblasts differentiation, which play a significant role as profibrotic agents in pterygium [28,29]. While EGF promotes excessive keratinization and the overexpressed receptor of EGF leaded to increased proliferation in pterygium epithelial cells [30,31]. So, we induced EMT in human conjunctival epithelial cells (HCEs) with TGF-β and EGF imitate the EMT initiation in pterygium.
This EMT pterygium cell model was applied to the subsequent experiments.
Our rationale was therefore to test whether miR-199a might provide new perspectives on pterygium mechanism in EMT and hope for a new and effective treatment.

Pterygium and control conjunctiva tissue samples
A total of 263 conjunctiva tissues were analysed in this study, including 234 pterygium and 29 control conjunctiva tissues. All pterygium samples were obtained from patients who diagnosed with primary pterygium undergoing a surgical resection and control conjunctiva tissues were collected from patients who underwent cataract surgery from December 2014 to October 2019 at Zhongnan Hospital of Wuhan University. Samples were stored at -80°C immediately after surgery until used.
This study was approved by the Medical Ethical Committee of Zhongnan Hospital of Wuhan University and followed the tenets of the Declaration of Helsinki and its later amendments. Informed consents were obtained from all patients before the study was carried out.

MiRNA microarray assay
Total RNA was isolated from frozen tissue using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. All RNA concentration and purity was evaluated by the NanoDrop2000 spectrophotometer (Thermo Scientific, MA, USA). Total RNA was purified and labelled by FlashTag™ Biotin HSR RNA Labelling Kit (P/N 901911, Affymetrix) according to the manufacturer's instructions to obtain biotin labelled miRNA. Array hybridization and wash was performed by GeneChip® Hybridization, Wash and Stain Kit (P/N900720, Affymetrix Santa Clara CA, USA) and GeneChip Eukaryotic Hybridization Control Kit (P/N 900454, Affymetrix Santa Clara CA, USA) in Hybridization Oven 645 (P/N 00-0331 (220V), Affymetrix Santa Clara CA, USA) and Fluidics Station 450 (P/N 00-0079, Affymetrix Santa Clara CA, USA) according to the manufacturer's instructions.
Arrays were scanned by GeneChip® Scanner 7G (Affymetrix, Santa Clara, CA, USA) and Command Console Software 3.2 (Affymetrix, Santa Clara, CA, USA) with default settings. Raw data was normalized by RMA and DABG algorithm, Expression Console (Affymetrix, Santa Clara, CA, USA).

RNA isolation, reverse transcription and quantitative real-time PCR
MiRNA and cDNA were synthesized from 500 ng total RNA by RevetAid RT Reverse Transcription Kit (Thermo Scientific, MA, USA) with specific miRNAs stem-loop RT primers (Table 1) and oligo d(T) 18 , using reverse transcription and quantitative real-time PCR (qRT-PCR). The miRNAs or mRNA expression levels were performed using Bio-Rad CFX96™ real-time PCR detection system (Bio-Rad, CA, USA) and each sample was detected in duplicate. Each assay consisted of 1 × SYBR Green quantitative real-time PCR master mix (Bio-Rad, CA, USA), 0.5 µM of forward and reverse primers (Table 1), and 1 µl cDNA template in a total volume of 20 µl. Non-template control was used as negative control, miRNAs reactions were normalized to U6, mRNA was normalized to GAPDH and the relative expression levels were calculated using the 2 −△Cq method. and 1uM penicillin-streptomycin (Gibco). Cells were grown at a humidified atmosphere of 5% CO 2 at 37°C. About 10 nM TGF-β and 20 nM EGF purchased were added after 24 h incubation in growth medium. The medium was changed every other day and TGF-β and EGF were reintroduced to main their concentration, and the cells that harvested after 7 days were used for subsequent experiments.
Immunofluorescence staining: For EMT markers validation, the treated HCEs were fixed with 3.5% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA, sigma, USA), and incubated over night at 4°C with primary antibodies as following: anti-N-

Wound healing and transwell assay
HConEpic dealt with with TGF-β and EGF for 7d, was seeded equivalently into 6-well culture plates and then was treated with transient transfection for 24 h before scratching or resuspending. A wound was scratched onto the mono-layer with a sterile 20 ul tip (Axygen, Union City, CA, USA). Images of HCEs migrating into the wound were captured at time points of 0, 24 and 48 h by an inverted microscope.
The migration assay were performed using upper chambers of Transwell insert (0.8um pore size, Corning Incorporated, Costar, USA) with 4*10 5 cells in serum-free medium for 48 h. After migration, cells passed through the coated membrane to the lower surface, where cell were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cell count was performed under a microscope.

Cell apoptosis determined with flow cytometry
The flow cytometry was performed to analyse the apoptosis of HCEs. Inducement or transfection was done before the cell re-suspended with Annexin V at the concentration of 10 6 cells/ml. Every specimen was incubated with Annexin V-FITC and propidiumiodide (PI) successively according to the manufacture's protocol (BestBio, China). Cell apoptosis was analysed via flow cytometry (FACSCanto II; BD Bioscience, Franklin Lake, NJ).

Western blot analysis
After cells were seeded in 6

Dual-luciferase reporter assay
The fragments of DUSP5 or MAP3K11 3'UTR whole region containing the wild-type binding sites of miR-199a -3p or miR-199a-5p, were amplified with primers containing restriction sites,

Statistical analysis
All data were analysed by GraphPad-Prism8.0 (Graph Pad, CA, USA) in independent t-test, Mann-Whitney U test or Pearson Correlation. p < 0.05 (two-tailed) was considered statistically significant.

Result
Expression profiling showed that miR-199a was upregulated in pterygium To identity miRNAs expression profiling in pterygium, miRNA microarray analysis was performed in 3 pairs of pterygium and control conjunctiva tissue. Of the 2578 miRNAs screened, 1362 were upregulated and 1216 were downregulated in pterygium compared with normal conjunctiva (Fig. 1a).
Using filtering criteria of p < 0.05 and log 2 Fold Change (FC) > 1, 40 differentially expressed miRNAs in pterygium tissues were selected (Fig. 1b). These differentially expressed miRNAs were listed (Table 3) and selected for heat map (Fig. 1c). Among them, 30 were upregulated and 10 were downregulated. Furthermore, a part of the results of the miRNAs microarray was verified in a small group of samples as the explorer category, including 55 pterygium and 12 control conjunctiva tissues with qRT-PCR. In line with results of miRNA microarray, the qRT-PCR demonstrated the same direction for these miRNAs except miR-675-5p, which presented no significant decrease in pterygium (p > 0.05) (Fig. 1d).
Based on the essential role of miR-199 family in the EMT process, and the primary exploration results of microarray, miR-199a-3p and miR-199a-5p were selected for further exploration on the potential mechanism downstream.

The EMT cell model was established using HCEs induced by TGF-β and EGF
We attempted to establish the EMT-pterygium cell model in HCEs, induced by TGF-β and EGF with different concentration gradients and time gradients. After cultured in the inducement of 10 nM TGF-β and 20 nM EGF for 7 days, the HCEs showed a distinct EMT phenotype (Fig. 3a). The characteristic of EMT-HCEs was identified with qRT-PCR, western blot and immunofluorescence. Compared with control HCEs without inducement, the expression of N-cadherin and Vimentin increased significantly, while Ecadherin decreased (Fig. 3b-3d). In order to confirm the migration ability of EMT activation, the wound healing and transwell assays were executed to find that TGF-β and EGF improved epithelial cells migration ( Fig. 3e and 3f). Furthermore, cell apoptosis was detected by flow cytometry with the Annexin V-FITC/PI reagent, and decreased apoptosis cells were observed after induced with TGF-β and EGF (Fig. 3g).
To further confirm that whether the EMT-HCE model could represent the EMT process in pterygium, we detected the expression of EMT markers in primary culture pterygium cells. We found that the primary culture cells of pterygium showed the same characteristics of EMT promotion as that of EMT-

HCE (Supplementary 2).
Knockdown miR-199a-3p/5p hindered EMT After the induced cells were transfected with miR-199a-3p and miR-199a-5p inhibitor respectively to inhibit the expression, features of EMT would be diminished according to the decreased EMT markers (Fig. 4a). The migration ability of EMT-HCEs was significantly increased, but decreased after treating with inhibitors (Fig. 4b). Our result further verified that the expression variation trend of miR-199a-3p or miR-199a-5p was consistent with the EMT process of HCEs.

DUSP5/MAP3K11 inhibition could promote EMT in HCEs
The effects of knockdown and overexpression were performed by qRT-PCR and western blot. The results showed that DUSP5-shRNA2# and MAP3K11-shRNA1# had the best knockdown effects, and both of the overexpression vectors demonstrate the effect of significantly upregulated expressions of the two gene ( Fig. 6a-6d). The HCEs were treated for 36 h for RNA extracting, 48 h for protein extracting, wound healing and cells migration (Fig. 6e and 6f). Both of the wound healing and transwell assay results showed that knockdown either DUSP5 or MAP3K11 promoted the HCEs migration, and when either of the two gene was overexpressed, EMT was inhibited ( Fig. 6g and 6 h).
The suppression on DUSP5/MAP3K11 induced by TGF-β and EGF could be regulated by miR-199a-3p/5p expression In the process of TGF-β and EGF induced EMT in HCE, the expression changes of miR-199a-3p, miR-199a-5p, DUSP5 and MAP3K11 were detected. To some extent, with the increase of TGF-β and EGF induction time, the EMT process was promoted, and the expression levels of miR-199a-3p and miR-199a-5p were also increased, while the expression levels of DUSP5 and MAP3K11 were decreased. WB results verified that, compared with HCE, the expression levels of DUSP5 and MAP3K11 of EMT-HCE were significantly reduced (Fig. 8a-8e). Inhibition of the expression of miR-199a-3p or miR-199a-5p in EMT-HCE resulted in increased expression of corresponding target genes (Fig. 8f-8i). That is to say, the inhibition of TGF-β and EGF on DUSP5 and MAP3K11 was weakened by decreasing the expression of miR-199a-3p and miR-199a-5p. We speculated that miR-199a-3p and miR-199a-5p may be involved in the occurrence of pterygium EMT by targeting DUSP5 and MAP3K11.
MiR-199a-3p/5p potentiated EMT induced by TGF-β and EGF by targeting DUSP5/MAP3K11 Transwell assays were then used to determine the effect of miR-199a-mediated target gene regulation on TGF-β and EGF-induced migration of HCEs. Treated with 10 nM TGF-β and 20 nM EGF for 7 days, the migration ability of HCEs was increased, while the effects were inhibited when either of DUSP5 or MAP3K11 was overexpressed. The migration ability of HCEs was enhanced when miR-199a-3p or miR-199a-5p was overexpressed by mimics, but decreased when either of the two miRNAs was knockdown by inhibitors ( Fig. 8a and 8b). The inhibition of miR-199a-3p or miR-199a-5p resulted in the enhancement of DUSP5 or MAP3K11-mediated inhibition of HCEs migration, and TGF-β and EGF induced migration of HCEs was increased by miR-199a-3p or miR-199a-5p treatment. The change trend of EMT markers was consistent with that of migration ability. Overexpression of DUSP5 or MAP3K11 rescued the effects of TGF-β and EGF-induced EMT, and miR-199a-3p and miR-199a-5p could further regulate the induction by regulating the expression of DUSP5 and MAP3K11 respectively ( Fig. 8c and 8d). All in all, inhibition of either of DUSP5 or MAP3K11 resulted in the promotion of EMT by miR-199a-3p or miR-199a-5p.
The expression level of MAP3K11 in tissues was low, so the results did not contain some meaningless value calculated by qRT-PCR. The amplification efficiency of these primers were verified (Supplementary 3). Downregulated expressions of DUSP5 and MAP3K11 proteins in pterygium tissues were further verified by western blot (Fig. 9e). Moreover, expression levels of miR-199a-3p and miR-199a-5p tended to be negatively associated with those of DUSP5 and MAP3K11 respectively (Fig. 9f and 9 g), which validated the previous experiments in HCEs.

Downstream pathway prediction by bioinformatics
In order to explore the possible roles of miR-199a-3p and miR-199a-5p in greater depth in the occurrence and development of EMT in pterygium, we subjected the putative targets of the two miRNAs to pathway enrichment analysis. The network showed that miR-199a-3p and miR-199a-5p might participate in the development of pterygium by affecting MAPK signalling pathway, TGF-β signalling pathway, PI3K-Akt signalling pathway, focal adhesion and others (Fig. 10a). From the pathway enrichment network, both of the DUSP5 and MAP3K11 were related with MAPK signalling pathway and we speculated that miR-199a might be involved in the MAPK pathway by regulating DUSP5 and MAP3K11, thereby promoting the EMT in HCE. Consistent with other researches, both of miR-199a-3p and miR-199a-5p had the effect on EMT processes and induced cell migration through MAPK signalling pathway, such as miR-199a-3p-DUSP5-ERK and miR-199a-5p-MAP3K11-JNK-p53 pathway. The pathway network was established based on our observation and bioinformatics analysis (Fig. 10b).

Discussion
The effects of miR-199a and their target genes DUSP5 and MAP3K11 on EMT, were characterized in our study, using HCEs and EMT-HCEs induced by TGF-β and EGF. In previous researches, both activated TGF-β and EGF participates in promoting the process of migration, proliferation and EMT in many different diseases, and there might even be some synergy between TGF-β and EGF [32][33][34]. On the other hand, EMT and related phenotypes, including migration promotion, extracellular matrix remodelling and apoptosis inhibition, could be regulated by the expression of miRNAs [35][36][37]. There was a relationship of mutual regulation between specific miRNAs and TGF-β. Our study indicated the feasibility of building EMT cell model with TGF-β and EGF, and presented that miR-199a-3p and miR-199a-5p participated in the process of EMT induced by TGF-β and EGF, by targeting DUSP5 and MAP3K11 in pterygium.
Firstly, we got the miRNA expression profile of pterygium from Affymetrix' miRNA 4.0 microarray and validated in a small sample of pterygium tissues, where we first indicated that miR-199a might promote the progression pterygium. The function and downstream pathway of miR-199a were performed in HCEs, and finally verified in a large sample of pterygium tissues. Although there were many researches, using microarray to detect the differentially expressed miRNAs in pterygium [19,2,20,18], the Affymetrix' miRNA 4.0 microarray provided more complete measurements of miRNA transcripts involved in gene regulation compared to GeneChip® miRNA 2.0 and 3.0, based on reports in Sanger miRBase 20.0 database. And For instance, a larger number of miRNA reads, reached to 6659, were discovered in our study compared to previous pterygium microarray research, such as Silin Chen's (GSE21346), only 1380 reads.
MiR-199 family, consisting of miR-199a/b-3p and miR-199a/b-5p, is encoded within the Dynamin (DNM) genes and exhibit high conservation across species [38]. There are mounting evidences suggesting that miR-199 family acts as a fatal effector of TGF-β signalling in many diseases, regulating multiple disordered processes including cell proliferation, apoptosis, migration, invasion and EMT [39,40,25]. The most typical biomarker change in EMT was the conversation from Ecadherin to N-cadherin, accompanied by the increase of cell migration and the decrease of cell apoptosis [41]. We found that as the time of treatment with TGF-β and EGF increased, expression levels of both miR-199a-3p and miR-199a-5p were increased gradually. And, the migration and EMT of EMT-HCEs could be suppressed by inhibiting the expression of either miR-199a-3p or miR-199a-5p. It disclosed that miR-199a-3p/5p were also acted as TGF-β effector in pterygium.
Recently, Antoon JW et al. found that inhibition of p38 mitogen-activated protein kinase (MAPK) signalling pathway even could reverse EMT [42], which is previously considered as a key process of inducting and maintaining the inflammation in various disease [43]. Accordingly, in our study both DUSP5 and MAP3K11 implicated in MAPK signalling pathway, might be a potential therapeutic agent targeted specifically to reverse EMT in pterygium. Knockdown DUSP5 or MAP3K11 promoted EMT.
Upregulated DUSP5 had been verified to inhibit the process of EMT in gastric cancer and hepatocellular carcinoma through MAPK pathway, and the cells showed a reduced migration ability and increased apoptosis to inhibit the progression of cancers [44,45]. Furthermore, combining with existing literature, our pathway bioinformatics analysis also speculated that DUSP5 and MAP3K11 function as a suppressor in cell EMT by downregulating ERK and JNK [46][47][48].
In accordance with the expression change of miR-199a under TGF-β and EGF induction, the expression level of DUSP5 and MAP3K11 was decreased with time gradient negatively. The results of compensation were shown that, the miR-199a promotion of migration and EMT was possibly regulated by the expression level of DUSP5 and MAP3K11. Regardless the effects of miR-199a-3p and miR-199a-5p, high expression of DUSP5 or MAP3K11 could also inhibit the EMT. That is to say, TGF-β and EGF affected the expression of DUSP5 and MAP3K11 through the regulation of miR-199a-3p and miR-199a-5p, so as to take part in the MAPK signaling pathway, further to promote the EMT of HCEs, and participate in the initiation and development of pterygium.
Of note, in the present study, the most worthy of mention were the establishment of the EMT-HCE model and exploration of miR-199a-DUSP5/MAP3K11-MAPK axis in the process of EMT in pterygium.
On the other hand, there were some limitations. Firstly, the number of control conjunctiva tissues was limited to obtain. Secondly, the investigation on large population in different stages of pterygium might be helpful for understanding miRNA's function in different stage of pterygium [6,17]. So, it was imperative to further expand the sample size in the followed-up study. Finally, the in vivo animal experiments were lack in the present study.

Conclusions
Our present research presented that TGF-b and EGF activated the miR-199a-DUSP5/MAP3K11-MAPK axis in the EMT process of pterygium. Our research results supplemented basic understanding of the were obtained from all patients before the study was carried out.

Consent for publication
Not application.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary Files
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