Hsa-miR-143-3p inhibits Wnt-β-catenin and MAPK signaling in human corneal epithelial stem cells

Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-β-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-β-catenin and MAPK signaling pathways. The expression of β-catenin, active β-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated.

In this study, limbal epithelial cells cultured by both 2D and 3D culture systems were used for the functional studies. The Real Architecture For 3D Tissue (3D RAFTs) are collagen-based hydrogels in which corneal stromal stem cells (CSSCs) are embedded, which serve as a model of corneal stromal stem cell niche 18 . Thus, the limbal epithelial cells cultured on the 3D RAFTs will have close interaction with CSSCs similar to its native environment. The epithelial or endothelial tissue equivalents produced by this technique are suitable for transplantation and used as a model for studying cellular interactions and functional characteristics 19,20 . Elucidating the signaling mechanism behind the regulation of CESCs by hsa-miR-143-3p will pave platform to develop new miRNA-based culture techniques for expanding the CESCs for transplantation and stem cell therapies to treat patients with limbal stem cell deficiency (LSCD).

Methods
Sample. The enucleated donor globes not suitable for transplantation and limbal rims received after corneal transplantation (donor age below 73 years) were obtained from Rotary Aravind International Eye Bank (Madurai, India), Moorfields Eye Hospital Lions Eye Bank (London, UK) and Veneto Eye Bank Foundation (Venice, Italy). The non-vascularized tissues with intact limbus were used in the study after thorough examination under stereo binocular microscope. Human donor tissues were handled in accordance with the tenets of the Declaration of Helsinki. The study was approved by Institutional Ethics Committee of Aravind Medical Research Foundation (RES2013038BAS) and the Moorfields Eye Hospital / UCL Institute of Ophthalmology Eye Tissue Repository (10/H0106/57-2011ETR10). Informed consent for the use of donor eyes for research were obtained from the legally authorized representatives.
Target prediction for hsa-miR-143-3p. The targets of hsa-miR-143-3p were predicted using miRWalk (Version 3.0) 21 and mirDIP (Version 4.1.1.6) 22 . The targets that were common in both miRWalk and mirDIP were selected to avoid false positives. The selected targets were subjected to analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping tool, Search pathway in KEGG mapper and grouped into functional categories. The targets associated with regulation of stem cells were selected for further analysis.
2D limbal epithelial cell culture. Limbal epithelial cells were cultured from the 2 mm limbal explants dissected from the donor globes or limbal rims. The explants were placed in the supplemented hormonal epithelial medium (SHEM) media 23 coated 35-mm cell culture dish (Nunc, Thermofisher Scientific, Massachusetts, United States) with epithelial side facing up. The explants were incubated at 37 °C for 20 min to allow for their attachment to the culture dish. The attached explants were then cultured in SHEM at 37 °C and 5% CO 2. The media was changed on alternate day till they reached 70 to 80% confluency.
Limbal epithelial cell culture on 3D RAFTs. 3D RAFTs containing CSSCs were used for the culture of primary limbal epithelial cells. The limbal epithelial cells were obtained by incubating the limbal rims in dispase II (2 mg/ml) for 45 min at 37 °C. Then the limbal epithelium was scraped off using a sterile scalpel. The total limbal epithelium was subjected to trypsin (0.25%) treatment for 30 min to obtain individual cells 24 .
For CSSC culture, the limbal region together with anterior stroma was dissected and subjected to collagenase-L (0.5 mg/ml) treatment for 16 h at 37 °C. The cells were separated from the solution through centrifugation and the pellet was resuspended in 3 ml of CSSC medium 25 . The resuspended cells were cultured at 37 °C in 5% CO 2 in fibronectin coated T25 flask for 24 h. The cells were supplemented with fresh CSSC medium after 24 h and the non-adhered cells were removed. By second day, selective trypsinization for CSSCs were carried out with 0.05% Trypsin-0.02% EDTA (Invitrogen) and the CSSCs were seeded in the fresh fibronectin coated T75 flask. The CSSCs were cultured at 37 °C in 5% CO 2 with media change on every alternate day. At 60-70% confluency, the cells were sub-cultured with the seeding density of 3000 cells/cm 2 in a fresh flask. For the preparation of 3D-RAFT, the CSSCs at passage 4, validated for the CSSC marker expression by immunostaining were used.
For the preparation of RAFT TEs, the AteloCell Native collagen (Bovine dermis, 3 mg/mL, pH 3.0, collagen acidic solution I-AC) (Koken, Tokyo, Japan) was mixed with 10X Minimum Essential Medium (MEM) from RAFT reagent kit (Lonza, Basel, Switzerland) in the ratio of 8:1. The collagen solution was adjusted to pH between 7.2 and 7.4 using the neutralising solution (5 M Sodium hydroxide) 26 . The solution was centrifuged at 1000 rpm for 3 min. The CSSCs (30,000 cells/ RAFT) were resuspended in the CSSC medium and added to the collagen solution. A volume of 625 µl of freshly prepared collagen solution with CSSCs was transferred to each well of a 24 well plate (Greiner, Stonehouse, UK) and heated to 37 °C for 30 min. Once the collagen gels were formed, RAFT absorbers for 24-well plates (hydrophilic porous absorbers) (Lonza) were applied to the surface of the hydrogels for 30 min. Then the absorbers were gently removed and fresh CSSC media was added to the RAFT TEs and stored at 37 °C. The limbal epithelial cells were seeded in 24 well plate at the density of 2.5 × 10 4 cells/ RAFT and cultured in SHEM media at 37 °C and 5% CO 2 . The cells were cultured until they reach 70 to 80% confluency with media change on every alternate day. miRNA transfection. The transfection of 70-80% confluent human primary limbal epithelial cell cultures grown in both 2D and 3D culture systems were carried out using Lipofectamine 3000 transfection reagent (Ther- Colony forming assay. The hsa-miR-143-3p mimic or inhibitor or scrambled control transfected cells were seeded on mitomycin C (4 µg/ml, Sigma-Aldrich) inactivated NIH 3T3 mouse fibroblast feeder layer in 60 mm dish independently at a seeding density of 500 cells per plate. After 12 days of culture in SHEM, the feeder layer was removed with 0.02% EDTA solution (Sigma-Aldrich) and the colonies in the dish were stained with 1% Rhodamine B (Roche, Basel, Switzerland) for 30 min after 15 min fixation with 4% paraformaldehyde (Sigma-Aldrich). Colonies were washed with sterile distilled water and images were captured (Nikon D750 camera, Japan). Colony forming efficiency (CFE) was calculated using the formula: number of colonies formed / number of cells seeded × 100. The colonies were termed as holoclone-like with respect to their size and morphology as defined by Barrandon and Green 27 . qPCR. The total RNA was isolated using RNeasy mini kit (Qiagen) from the three groups (i) mimic treated, Immunofluorescence staining. The transfected cells grown in 2D culture system were trypsinised with TrypLE Express (Gibco-Thermofisher Scientific) after 2 days of culture and cytocentrifuged (400 rpm; 3 min) on the slides (2.5 × 10 4 cells/slide). The cells were fixed for 20 min with 4% paraformaldehyde at 25 °C. After fixation, the cells were washed with 1X PBS (thrice) and permeabilized with 0.5% Triton X-100 for 10 min at 25 °C. Following permeabilization, cells were washed with 1X PBS and blocked with 5% goat serum (Sigma-Aldrich) for 60 min. The cells were then incubated with primary antibody diluted in 2% goat serum at 4 °C over night (Supplementary Table S2: List of antibodies used for immunostaining). The cells were washed with PBS to remove the unbound antibodies and incubated with appropriate secondary antibody (1:500) conjugated with fluorophore (Alexa Fluor 488 or Alexa Fluor 555) in PBS for 60 min at 25 °C. The immunostained cells were mounted using vectashield medium with DAPI (Vector laboratories Ltd, Peterborough, UK) after thorough washing with PBS and sealed with coverslip. The images were acquired using confocal laser scanning microscope (Zeiss LSM 700, Germany) for analyzing the localization pattern of protein expression. For each primary antibody used, the corresponding isotype control was used as negative control. The experiment was replicated thrice with three biological samples (n = 3). The transfected cells on 3D RAFTs were immunostained directly on the culture plates and they were not subjected to trypsin treatment. For quantification of the protein expression, the images were taken with the fluorescence microscope Axioskop 2 (Zeiss). The marker expression was quantified with histogram function of HCImage analyser software based on the mean intensity of the fluorescence signal in different channels. The relative protein expression based on fluorescence intensity was quantified as described by Lee et al. 28 Western blotting. For the isolation of protein from the transfected cells, the cells were lysed with the mixture of RIPA lysis and extraction buffer (Thermo Scientific) and Halt protease inhibitor cocktail (Thermo Scientific) after washing with ice cold PBS (Gibco, Thermo Scientific). The concentration of the isolated protein was estimated using BCA Protein Assay kit (Pierce, Thermo Scientific). Equal amounts of extracted protein from each sample (20 µg) were separated by 10% Bis-tris gel (NuPAGE, Thermo Scientific) under reducing conditions after heating for 10 min at 95 °C along with LDS sample-loading buffer (Thermo Scientific). The separated proteins were then electro transferred to a PVDF membrane (Invitrolon, Thermo Scientific). The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated at 4 °C overnight with primary antibody (Ab) (Supplementary Table S3: List of primary antibodies used for Western blotting). The membranes were washed thrice and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at 25 °C for 1 h (Cell Signaling Technology, Inc., Massachusetts, United States). The protein bands were detected using enhanced chemiluminescence reagent after thorough washing with TBST (Millipore, Billerica, MA). In every blot, GAPDH was used as normalizing reference and loading control. The experiment was repeated thrice using the limbal epithelial cells grown both in 2D and 3D culture system and the data were represented as the mean ± SD. To analyze multiple proteins from the same blot and to avoid repeated stripping, the membrane was cut based on the molecular weight of the protein to be analyzed, prior to hybridization with antibodies. Statistical analysis. Statistical software STATA 14.0 (Texas, USA) was used for the statistical analysis. All the experiments were carried out in triplicates and the data were represented as mean ± SD. Independent t-test www.nature.com/scientificreports/ (parametric) was performed to compare the two experimental group when the data followed Gaussian distribution and Mann-Whitney U test (non-parametric) was used for the data that followed non-Gaussian distribution based on the Shapiro-Wilk normality test. p < 0.05 was considered to be statistically significant.

Regulation of stemness and differentiation by hsa
The Western blot analysis of stem cell markers and differentiation marker in the transfected cells reavealed that the level of expression of ABCG2 (2.14 ± 0.47, p = 0.0134) and ΔNp63α (2.07 ± 0.37, p = 0.0074) were upregulated and Cx43 expression (0.23 ± 0.05, p < 0.0001) was downregulated in mimic transfected cells (Fig. 2).
To confirm the presence of stem cells in transfected cultures, colony forming assay was carried out. The mimic treated cells showed increased percentage of colony forming efficiency (10.04 ± 0.45, p = 0.0003) compared to that of control (3.33 ± 0.23) and inhibitor treated cells (0.26 ± 0.08, p = 0.0003). In addition, there was a significant increase in the percentage of holoclone-like colonies (7.66 ± 2.45, p < 0.05) compared to that of the control (0.69 ± 2.08). The inhibitor treated cells produced no such larger colony (Fig. 4). Thus, the higher expression of hsa-miR-143-3p increased the colony forming efficiency and supported holoclone-like colony formation.

Discussion
Regulatory role for miRNAs has been identified in various stem cells recently. MiRNAs modulate signaling pathways through selective targeting of the molecules involved 30,31 . For the maintenance of hematopoietic stem cells, miR-143 downregulated Smad-dependent TGFβ/DAB2 signaling 32 . In bone marrow mesenchymal stem cells and apical papillary stem cells, miR-143-3p negatively regulated the differentiation process through targeting bone morphogenetic protein 2 33 and nuclear factor I-C 34 respectively. In contrast, it supported differentiation in human dental pulp stem cells via targeting Osteoprotegerin-RANK signaling pathway 35 . Though the expression of hsa-miR-143-3p was identified in various ocular tissues 11,36-38 , its functional association has not been explored. www.nature.com/scientificreports/ Our previous study on miRNA profiling of enriched CESCs 9 suggested the possible role of hsa-miR-143-3p in the maintenance of stemness in CESCs. In continuation, the ectopic expression of hsa-miR-143-3p in this study, increased the colony forming potential of cultured limbal epithelial cells along with increased number of holoclone-like colonies based on the size and morphology. In addition, the expression of stem cell markers was  Table S4 and original Western blots images are provided in Fig. S2.    Table S5 and original Western blots images are provided in Fig. S4 [43][44][45] . The regulatory role of hsa-miR-143-3p's predicted targets in Wnt and MAPK signaling has been tabulated ( Table 1). The expression of Wnt signaling regulators: AXIN2, β-catenin, active β-catenin and MAPK signaling regulators:p-ERK1/2, p-p38, p-c-JUN, p-c-FOS, p-ATF2, p-p53 were downregulated in mimic transfected cells indicating the inhibition of Wnt-β-catenin and MAPK signaling. Based on the above observations, we hypothesized a probable mechanism of action of hsa-miR-143-3p on Wnt and MAPK signaling which has been summarized in Fig. 7.
Hsa-miR-143-3p inhibits MAPK signaling by downregulating the expression of its targets (i) MAPK14activation of MAPK14 is the essential step and which marks the activation of p38 MAPK signaling, (ii) DVL3initiates JNK MAPK signaling through activation of RAC protein, (iii) MAPK1-activation and translocation of MAPK1 to the nucleus is the crucial step in the activation of ERK MAPK signaling and (iv) KRAS-activates ERK MAPK signaling through activation of RAF protein.

Conclusion
The probable association of hsa-miR-143-3p in the maintenance of CESCs through downregulation of key proteins involved in Wnt and MAPK signaling has been demonstrated in this study using primary limbal epithelial cells grown in 2D and 3D culture systems. Further functional studies are essential to confirm whether the predicted genes are the direct targets of hsa-miR-143-3p and their role in the maintenance of stemness.