microRNA-103/107 family regulates multiple epithelial stem cell characteristics

Han Peng; Jong Kook Park; Julia Katsnelson; Nihal Kaplan; Wending Yang; Spiro Getsios; Robert M Lavker

doi:10.1002/stem.1962

. Author manuscript; available in PMC: 2016 May 1.

Published in final edited form as: Stem Cells. 2015 May;33(5):1642–1656. doi: 10.1002/stem.1962

microRNA-103/107 family regulates multiple epithelial stem cell characteristics

Han Peng ^1,^*, Jong Kook Park ^1,^*, Julia Katsnelson ^2,^*, Nihal Kaplan ¹, Wending Yang ¹, Spiro Getsios ¹, Robert M Lavker ¹

PMCID: PMC4409488 NIHMSID: NIHMS658893 PMID: 25639731

Abstract

The stem cell niche is thought to affect cell cycle quiescence, proliferative capacity and communication between stem cells and their neighbors. How these activities are controlled is not completely understood. Here we define a microRNA family (miRs-103/107) preferentially expressed in the stem cell-enriched limbal epithelium that regulates and integrates these stem cell characteristics. miRs-103/107 target the ribosomal kinase p90RSK2, thereby arresting cells in G0/G1 and contributing to a slow-cycling phenotype. Furthermore, miRs-103/107 increase the proliferative capacity of keratinocytes by targeting Wnt3a, which enhances Sox 9 and YAP 1 levels and thus promotes a stem cell phenotype. This miRNA family also regulates keratinocyte cell-cell communication by targeting: (i) the scaffolding protein NEDD9, preserving E-cadherin-mediated cell adhesion; and (ii) the tyrosine phosphatase PTPRM, which negatively regulates connexin 43-based gap junctions. We propose that such regulation of cell communication and adhesion molecules maintains the integrity of the stem cell niche ultimately preserving self-renewal, a hallmark of epithelial stem cells.

Keywords: cell cycle, proliferative capacity, Wnt signaling, adherens junctions, gap junctions, cell-cell communication

Introduction

As a consequence of the vast amount of work focused on the biology of adult epithelial stem cells conducted over the past 35 years, a set of criteria has been established defining these progenitor cells. In its simplest form, a stem cell must have unlimited division potential and give rise to progeny that are also capable of divisions but in a more finite manner [1]. Additional features associated with epithelial stem cells are quiescence [2], and a primitive, undifferentiated phenotype [3, 4]. Another aspect of adult epithelial stem cells is their non-random distribution with a particular epithelium. For example, in the small intestine, stem cells are positioned in lower portion of the crypt [1], esophageal stem cells are localized in the interpaillary zone [5], corneal epithelial stem cells are localized to limbal epithelium [4, 6, 7], hair follicle stem cells reside in the “bulge” region of the outer root sheath [8], and adult epidermal stem cells are believed to be located at the tips of the rete ridges [3, 9, 10], although this is still being debated [11, 12]. Such clustering of stem cells is evidence for existence of a “niche”, which regulates stem cell behavior [13-16].

The stem cell niche varies depending on the epithelial tissue type but generally consists of a group of heterogeneous cells (stem cells and their transit amplifying (TA) progeny) that are anchored together by specific junctions. In addition, this cluster of cells interfaces with the basement membrane and communicates with the immediately subjacent extracellular matrix [17]. Jointly, such interactions preserve stem cell function, which is to self-renew and give rise to more differentiated progeny [15, 16]. With respect to stratified squamous epithelia and their appendages, it is becoming apparent that communication between stem cells and their progeny plays a greater role in regulating aspects of stem cell behavior than was previously appreciated [18-20]. However, it is unclear how the cell-cell contacts within the niche are regulated and how this impacts on stem cell characteristics such as quiescence and proliferative capacity.

microRNAs (miRNAs), a critical part of the RNA silencing machinery, are known to play important regulatory roles in stem cell biology. For example, disruption of the dicer-1 gene in mouse embryonic stem cells (ES) resulted in defects in: (i) division and proliferation; and (ii) differentiation both in vitro and in vivo. Furthermore, loss of Dicer 1 in mice resulted in embryonic lethality due to a depletion of stem cells [21]. In neuronal stem cells, miR-184 induces proliferation and inhibits differentiation [22], while miR-124, plays a role in inducing NSC differentiation [23]. Throughout ESC differentiation, muscle-specific miRs-1 and −133 suppress nonmuscle gene expression during cell lineage commitment and regulate cell-fate from pluripotent ESCs [24]. miR-499 is thought to induce rat bone marrow-derived mesenchymal stem cells (MSCs) to differentiate into cardiac cells via the Wnt/β-catenin signal pathway [25]. MSC differentiation towards a chondrocyte lineage is believed to be under the direction of miRs-152,-28-26b and −193b [26]. With respect to epithelial stem cells, miR-205 is one of several miRNAs that are highly expressed in mammary progenitor cells [27]. Interestingly, only two miRNAs have been associated with the regulation of adult epidermal and/or appendageal stem cells. miR-203 has received a great deal of attention as it targets p63, a putative regulator of epithelial stem cell-maintenance and differentiation [28, 29]. More recently, miR-125b has been reported to target Blimp1, thereby helping to maintain epithelial stem cells in a hair follicle lineage [30].

To identify miRNAs that are associated with an epithelial stem cell phenotype, we have chosen to use the corneal/limbal epithelia as a paradigm as this system is ideally suited for studies on epithelial stem cells for the following reasons: (i) Corneal epithelial stem cells are primarily located in the basal layer of the limbal epithelium [4, 6, 31]. Such an anatomical location results in a distinct physical separation between the limbal epithelial-enriched stem cells and their corneal epithelial-enriched TA cell progeny. This physical separation enables the isolation of relatively pure populations of stem versus TA cells for biochemical and physiological investigations [32]; (ii) Limbal epithelial stem cells share properties that are commonly associated with stem cells from other self-renewing tissues [33]. They cycle infrequently [6], are relatively undifferentiated [4], have a high in vivo and in vitro proliferative potential [34] and can be used to reconstitute the entire corneal epithelium [35, 36]; and (iii)The corneal/limbal system is one of the “poster children” for regenerative medicine, where ex vivo expansion of the limbal epithelial stem cell pool generates sheets of epithelium that are subsequently used clinically for corneal epithelial transplants [36, 37].

In the present study, we demonstrate that the miRs-103/107 family are preferentially expressed in the basal cells of the stem cell-enriched limbal epithelium and target novel proteins involved in processes related to stem cells. We show that miRs-103/107 target p90RSK2, a kinase that regulates G0/G1 progression and in this manner helps maintain a slow-cycling phenotype. The proliferative capacity of human epidermal and limbal keratinocytes is enhanced by ectopic expression of the miR-103/107 family, in part, by targeting Wnt3a. By targeting NEDD9 (HEF1), miRs-103/107 insures maintenance of the essential stem cell niche molecule, E-cadherin (E-cad) in limbal keratinocytes. We also demonstrate that the tyrosine phosphatase PTPRM is a target of miRs-103/107 and thereby maintains low levels of Cx43, which is a feature of several stem cell-enriched epithelia. Collectively these findings indicate that miRs-103/107 play critical roles in orchestrating multiple aspects of the adult epithelial stem cell.

Material and Methods

Mice

Wild-type mice (Balb/c) were obtained from the Charles River Laboratories. Mice were killed by CO2 asphyxiation and cervical dislocation. Whole globes with surrounding tissues were excised, immediately embedded in OCT compound and stored at −80 °C or in paraffin. Animal experiments were approved by the Northwestern University Animal Care and Use Committee (NUACUC).

Cell culture, transduction and transfection

Primary cultures of HEKs were isolated from neonatal foreskin by NU SDRC keratinocyte core and maintained in medium 154 (Cascade Biologics) containing HKGS growth supplements and 0.07mM CaCl₂. Primary HCEKs and HLEKs were isolated from cadaver donor corneas provided by Midwest Eye Bank (Ann Arbor, MI) and cultured in CnT-20 media with supplements (CellnTech) on collagen IV-coated plates (BD Biosciences). The limbal derived corneal epithelial cell line, hTCEpi, was grown in Keratinocyte SFM medium with supplements (Invitrogen) and 0.15mM CaCl₂. For Wnt3a treatment, keratinocytes were plated on collagen IV-coated plates with CnT-20 media and grown overnight. Recombinant wnt3a (R&D system) were added in several concentrations for 24 hours. For lentiviral infections, keratinocytes were transduced with lentiviral supernatants (produced by the NU-SDRC RNA/DNA Delivery Core Facility) for 6 hours and switched to fresh culture medium overnight. To silence gene expression, siRNA Smart pools (20 nM) targeting p90RSK2, Nedd9, PTPRM or a scrambled negative control (Dharmacon, Lafayette, CO) were transiently transfected into cells using siRNA transfection reagent (RNAiMAX; Invitrogen) as described [38]. A cDNA encoding miRs-103/107-resistant Nedd9 was ligated into the lentiviral expression plasmid pCDH (System Biosciences). The following constructs were used in this study: Human premicroRNA Expression Construct Lenti-miR-107 and Human premicroRNA Expression Construct Lenti-miR-103 (System Biosciences).

Antagomirs

Antagomirs were synthesized by Dharmacon. Sequences were 5’- mU(*)mC(*)mA(*)mUmAmGmCmCmCmUmGmUmAmCmAmAmUmGmCmU(*)mG(*)mC(*)mU - Chol-3’ (antagomir-103), 5’-mU(*)mG(*)mA(*)mUmAmGmCmCmCmUmGmUmAmCmAmAmUmGmCmU(*)mG(*)mC(*)mU-Chol-3’ (antagomir-107), 5’- mG(*)mG(*)mC(*)mAmUmUmCmAmCmCmGmCmGmUmGmCmC(*)mU(*)mU(*)mA-Chol-3’ (irrelevant-antagomir). “mN” represents 2’-Omethyl-modified oligonucleotide, the “(*)” represents a phosphorothioate linkage, and “Chol” represents linked cholesterol.

Laser capture microdissection and microRNA profiling

Eyes from wild-type mice (Balb/c) were embedded in optimal cutting temperature (OCT) compound and stored at −80 °C until sectioning. Basal cells from limbal and corneal epithelia from 5 μm frozen sections were isolated and captured using a PALM laser capture system (Zeiss) as previously described[39]. Total cellular RNA from LCM-captured cells was isolated and purified by miRNeasy kit (Qiagen). microRNA profiling was performed using Exiqon's miRCURY LNA Universal RT miRNA PCR Rodent panel I and II by Exiqon. Briefly, 5 ul RNA was reverse transcribed in 40μl reactions using the miRCURY LNA™ Universal RT microRNA PCR, Polyadenylation and cDNA synthesis kit (Exiqon). cDNA was diluted 100 × and assayed in 10 ul PCR reactions according to the protocol for miRCURY LNA™ Universal RT microRNA PCR; each microRNA was assayed once by qPCR on the microRNA Ready-to-Use PCR, Rodent panel I and panel II. Negative controls excluding template from the reverse transcription reaction was performed and profiled like the samples. The amplification was performed in a LightCycler® 480 Real-Time PCR System (Roche) in 384 well plates. The amplification curves were analyzed using the Roche LC software, both for determination of Cp (by the 2nd derivative method) and for melting curve analysis.

mRNA expression profiling

Total cellular RNAs from antagomir-103,-107 and −124 (irrelevant control)-treated primary human limbal epithelial keratinocytes were isolated and purified using an mRNeasy kit (Qiagen, Hilden, Germany). RNA quality (all samples had RIN values >9.1) was verified using an Agilent Bioanalyser 2100 (Agilent, Santa Clara, CA, USA). Expression profiling was performed using an Illumina HumanHT-12 kit (Illumina, Inc.). All the samples met the Illumina standard quality control checks. Data quality checks were performed using a Bioconductor Lumi package for R statistical programming environment. The data processing also included a normalization procedure utilizing quantile normalization method to reduce the obscuring variation between microarrays. The MetaCore online tool (Thomson Reuters) was used for Gene Ontology enrichment analysis.

In situ hybridization

In situ hybridization was performed as described previously [40]. 20nM locked nucleic acid (LNA)-modified digoxigenin (DIG)-labeled probes (Exiqon) against miR-103 and miR-107 were used.

Cell cycle analysis

For G0/G1 synchronization, cells were maintained for 24 h without supplements. Cells were then grown with supplements for 3 hours. Cells were trypsinized, harvested, and fixed in 1 ml 80 % cold ethanol in test tubes and incubated at −20 °C for 2 hours. After incubation, cells were centrifuged at 1,500 rpm for 5 min and the cell pellets were resuspended in 500 μl propidium iodine (50 μg/ml) containing 200 μg/ml RNase A (Sigma). Then cells were incubated at 37 °C for 20 min and filtered with 53 μm nylon mesh. Cell cycle distribution was calculated from 30,000 cells using FACSCalibur Flow Cytometer (BD Biosciences).

Western Blotting

Western blots were performed as described previously[39]. The following antibodies were used: p-FAK(Y925), p90RSK2, p-p90RSK (S380), p-p90RSK (T359/S363), connexin43, NEDD9, p-Src, Src, p-JNK(Cell Signaling Technologies), p-FAK (Y576/577), Wnt3a, PTPRM, GAPDH (Santa Cruz Biotechnology), p120-catenin (BD Biosciences), E-cadherin (TaKaRa Bio), SOX9 (Abcam), PTPRM (CHEMICON). To detect secreted wnt3a from antagomiR treated cells, equal volumes of cell culture media were concentrated using Protein Concentrator, 9K MWCO (Thermo Scientific Pierce) and separated by SDS-PAGE using standard methods.

Immunofluorescence and Immunocytochemistry

Normal human corneas were obtained from the Illinois Eye Bank. Frozen sections (5μm) were processed for fluorescence immunostaining as described previously[39]. Briefly, sections were incubated for 1h with primary antibodies against E-cadherin (1:10; TaKaRa Bio) or connexin 43(1:50; Cell Signaling Technologies). DAPI was used to counterstain nuclei. Immunocytochemistry was performed as previously described [39]. Briefly, cells were fixed with 4% PFA for 10min. Slides were incubated overnight with an antibody recognizing E-cadherin (1:10; TaKaRa Bio) or connexin 43(1:50; Cell Signaling Technologies ) DAPI was used to counterstain nuclei. For BrdU staining to detect proliferation, cells were treated with 10 μM BrdU for 1 h and then the media was replaced and incubated for an additional hour before fixing in methanol at −20 °C for 10 min. Antigen retrieval was performed at 70 °C in formamide retrieval solution (1× SCC in formamide) for 30 min. After blocking in PBS containing 0.01% BSA and 0.01% Tween-20, slides were incubated for 30 min with BrdU monoclonal antibody (1:10; Developmental Studies Hybridoma Bank). After washing, slides were incubated with Alexa 555-linked secondary antimouse IgG (Vector). DAPI was used to counterstain nuclei. Images were acquired on an epifluorescence microscope system (AxioVision Z1; Carl Zeiss) fitted with a digital camera (AxioCam MRm; Carl Zeiss).

Colony Formation assay

As previously described[41], HLEKs and HEKs were plated in FAD medium (DMEM/F12 media, FCS (10%), insulin (5 μg/ml), adenine (0.18 mM), hydrocortisone (0.4 μg/ml), cholera toxin (10ng/ml), triiodothyronine (5μg/ml), 5ug/ml Human apo-transferrin, glutamine (4 mM), penicillin-streptomycin (50 IU/ml), and Epidermal growth factor (10 ng/ml)) in the presence of mitomycin C-treated 3T3 feeder cells at 100 cells per 100mm dish. To test whether Wnt3a treatment can inhibit colony formation, HEKs were plated in FAD medium with or without Wnt3a (30ng/ml; R&D system) at 200 cells per 100mm dish. After 2 weeks, the cells were fixed with methanol and stained with crystal violet.

Progenitor competition assay

HEKs were transduced with either a GFP-tagged miR −103 or miR-107 construct or a GFP-tagged scrambled construct. Equal numbers of GFP-tagged and untagged HEKs were mixed and allowed to form 3-D raft cultures as described previously [39]. The raft cultures were embedded in OCT and frozen sections were fixed in 4% PFA and then washed with PBS. DAPI was used to counterstain nuclei. Images were acquired on an epifluorescence microscope system (AxioVision Z1; Carl Zeiss) fitted with a slide module (Apotome; Carl Zeiss) and a digital camera (AxioCam MRm;Carl Zeiss).

Real time qPCR

Total RNAs were isolated by miRNeasy kit (Qiagen) according to the manufacturer's instructions. Taqman microRNA Assays (Applied Biosystems) was performed according to the manufacturer’s instructions. To detect mRNA levels, cDNA was prepared using SuperScript III reverse transcription kit (Invitrogen). Realtime qPCR was performed on an Applied Biosystems 7000 Real-Time PCR system using the Quantitative SYBR green PCR kit (Qiagen) according to the manufacturer’s instructions. Primer sequences used in this study were designed by IDT(Supporting information Table S4).

Inhibitors

For pharmacological inhibition of protein tyrosine phosphatases, cells were treated with 50 uM Sodium Orthovanadate (Vanadate, New England BioLabs) with and without treatment of Ir-antago, antago-103 or antago-107.

Scrape loading dye transfer assay

Following treatments, cells on coverslips were rinsed three times with calcium and magnesium free DPBS and covered with 1mg/ml Lucifer yellow CH solution in DPBS. Scrape were made with a metal blade and cells were kept at room temperature for 10 min. Coverslips were extensively rinsed several times with DPBS followed by fixation with 4% PFA for 10min and examined under an epifluorescence microscope system (AxioVision Z1; Carl Zeiss) fitted with a digital camera (AxioCam MRm; Carl Zeiss).

Luciferase Reporter Constructs and Assay

The three prime untranslated region (3’UTR) of miR-103/107 predicted targets was PCR amplified using human genomic DNA as a template and a LongAmp™ TaqDNA Polymerase (New England BioLabs). The PCR amplicons were subsequently gel purified, and ligated into the psiCHECK-2 vector (Promega). The mutant reporter constructs harboring mutation of the first three nucleotides of the seed-match sequence were constructed using the QuikChange site-directed mutagenesis kit (Stratagene). Schematic diagram of miR-103/107 binding sites and primers are provided in Figure S11 and Table S2, respectively. Screening the interaction the target genes with miRs-103/107 using the psiCHECKTM-2 vector harboring a wild or mutant 3’ untranslated region (UTR). Constructs were co-transfected with either miR-control, miR-103 or miR-107 into cells. Twenty four hour after transfection, cell lysates were used to measure both firefly and renilla luciferase activities using the Dual-Luciferase® Reporter Assay System (Promega) following the manufacturer's instructions.

Statistical analysis

All values are expressed as mean ± SD. The significance of the differences between 2 groups was evaluated by an unpaired Student’s t test.

Results

miR-103/107 family is limbal epithelial-preferred

The distinct physical separation between the limbal and corneal epithelium enabled us to isolate relatively pure populations of mouse limbal (stem cell-enriched) and corneal epithelial (TA-enriched) basal cells using laser capture microdissection [32]. We obtained miRNA expression profiles from the isolated limbal and corneal epithelial basal cells. Whereas most miRNAs were not preferentially expressed, the miR-103 and miR-107 family (miRs-103/107) were limbal epithelial-preferred (Supporting Information Table1; Supporting Information Fig. S1A) as confirmed by in situ hybridization (Supporting Information Fig. S1B). We also determined the expression pattern of miRs-103/107 in mouse epidermis via in situ hybridization and detected a strong signal for both miRNAs throughout the viable epidermis (Supporting Information Fig. S1B).

miR-103/107 family arrests keratinocytes in G0/G1

Since slow or infrequent cycling is a criterion for adult stem cells [14, 33, 42, 43] and miRs-103/107 are preferentially expressed in the stem cell-enriched limbal epithelium, we explored the possibility that miRs-103/107 altered the keratinocyte cell cycle. We first analyzed the effects of elevating miRs-103/107 levels by greater than 2-fold in primary cultures of human corneal epithelial keratinocytes (HCEKs), since in vivo these transient amplifying cells normally expressed low levels of miRs-103/107 (Supporting Information Figs. S1 A and B; Supporting Information Fig. S2A). Two weeks following transduction of this miRNA family, there were 60% and 80% fewer miR-103 and miR-107 transduced HCEKs, respectively, compared with the empty vector (pCDH) transduced HCEKs (Fig. 1A). A similar reduction in the number of cells following transduction of miRs-103/107 were noted for primary human limbal epithelial keratinocytes (HLEKs) and a limbal-derived human epithelial cell line (hTCEpi; [44]; Figs. 1B, C). The proliferative status of the transduced keratinocytes was also altered as evidenced by a significant reduction (*p<0.05) in the number of HCEKs, HLEKs, primary human foreskin keratinocytes (HEKs) and hTCEpi cells in the “S” phase of the cell cycle (Figs. 1D-G). To test further that ectopic expression of miRs-103/107 altered proliferation, we transduced an aggressive oral (tongue) squamous cell carcinoma cell line (CAL27) [45] and observed a significant increase in the population doubling time (Supporting Information Fig. S2B). Together our findings demonstrate that miRs-103/107 attenuate cell growth in normal and transformed keratinocytes from several stratifying epithelia.

miR-103/107 family regulates keratinocyte cell kinetics. (A) Cell growth in HCEKs transduced with an empty vector (pCDH), miR-103, or miR-107 was analyzed by crystal violet staining. Cellularity was measured using Image J. Data are shown as mean ± SE (N=3).*p<0.05. (**B, C**) Cell proliferation in HLEKs (B) and hTCEpi (C) cells transduced with a miR-negative control, miR-103, or miR-107 was analyzed by the WST-1 proliferation assay. Data are shown as mean ± SE (N=3).*p<0.05. (D-G) Bromodeoxyuridine (BrdU) analysis of HCEKs (D), HLEKs (E), HEKs (F), and hTCEpi cells (G) transduced with an empty vector (pCDH), miR-103, or miR-107 showing cells in the S phase of DNA synthesis. (H) Cell cycle distribution of hTCEpi cells transduced with an empty vector (pCDH), miR-103, or miR-107 was analyzed by flow cytometry, showing that miR-103/107 induced a G1 phase arrest.

Decreases in cell number, BrdU incorporation and increased population doubling times, suggest an arrest in the cell cycle, thus we used FACS analysis to investigate the cell cycle kinetics of hTECpi cells transduced with miR-103 or miR-107. We used this human limbal-derived keratinocyte cell line as it is easier to synchronize than primary human keratinocytes. Following transduction, cells were synchronized by removing supplemental growth factors and FACS sorted 3 hr after re-addition of the supplemental media (Fig. 1H). After the addition of the supplemental medium, 65% of the control transduced cells were in G0/G1, 22% were in S and 13% were in G2/M (Fig. 1H). In contrast, ~85% of miRs-103/107 transduced cells were in G0/G1, 5% were in S and 10% were in G2/M (Fig. 1H). Thus, ectopic expression of miRs-103/107 arrests keratinocytes in the G0/G1 phase of the cell cycle.

miR-103/107 family targets p90RSK2

miR-103 and miR-107 belong to the miR-103/107 family, have identical sequences except for one nucleotide at the 3’-end and regulate overlapping targets (Trajkovski et al., 2011; Chen et al., 2012;Zhang et al, 2012). Therefore we used in silico and cell-based luciferase assays to find common targets which regulate the G0/G1 phase of the cell cycle and found that p90RSK2 was a novel target of this miRNA family (Figs. 2A and B). We demonstrated that p90RSK2 was a bona fide target of miRs103/107 by their ectopic expression in hTCEpi cells (Fig 2C). We also used an antagomiR approach (Supporting Information Figs. S4 A-C) to knock down miRs-103/107 in HLEKs and hTCEpi cells and noted a significant increase in p-p90RSK levels compared to antago-124, an irrelevant antagomir specific for neuronal tissues [46] (Fig. 2D).

We then determined whether p90RSK2 regulated proliferation in keratinocytes. Treatment of HLEKs and hTCEpi cells for 48h with Bi-D1870, a p90RSK inhibitor [47], resulted in a 65% reduction in cell number (Fig. 2E). Similarly, gene silencing of RSK2 led to a marked reduction of p-p90RSK in hTCEpi cells (Fig. 2F). This resulted in a reduction in cell numbers in HLEKs and hTCEpi cells (Fig. 2G), a 40% reduction in BrdU incorporation by hTCEp1 cells (Fig. 2H) and a 15% increase in the population doubling time of hTCEpi cells (Supporting Information Fig. S3). The cell cycle kinetics of hTCEpi cells treated with sip90RSK2 (Fig. 2I) was similar to the ectopic expression of miRs-103/107 in these cells (Fig. 1H), with the knockdown of p90RSK2 resulting in an increase in cells in G0/G1 and fewer cells in S and G2/M when compared with the control siRNA. Therefore, we propose that miRs-103/107 contribute to a slow cycling phenotype by attenuating p90RSK, which holds these cells in G0/G1.

miR-103/107 family maintains keratinocytes in the basal layer

To place the cell kinetic data obtained from primary human epithelial cell cultures into a more physiologically relevant context, we utilized a three-dimensional (3-D) organotypic raft model of human epidermis [39, 48]. In this system, we transduced HEKs with a GFP-tagged miR-107 construct or a GFP-tagged empty vector construct. Equal numbers of GFP-tagged and untagged HEKs were mixed and allowed to form 3-D raft cultures (Fig. 3A). In the GFP-empty vector/untagged HEK-derived rafts the ratio of GFP-tagged to untagged basal cells was 50% (Fig. 3B), whereas in the GFP-miR-107/untagged HEK-derived rafts, the ratio of tagged to untagged cells increased to 80% (Fig. 3B). The persistence of miR-107-expressing cells in the basal layer is consistent with a slow-cycling, kinetically-quiescent cell (Fig. 1); features associated with stem cells [6, 8, 19]. This raises the question of whether ectopic expression of the miR-103/107 family enhances the proliferative capacity of keratinocytes.

miR-103/107 family confers progenitor cell status to keratinocytes. (A) Progenitor competition assay in three-dimensional (3-D) organotypic raft model of human epidermis. HEKs were transduced with either a GFP-tagged miR-107 construct or a GFP-tagged scrambled construct. Equal numbers of GFP-tagged and untagged HEKs were mixed and allowed to form 3-D raft cultures. The insets show GFP positive cells within boxed area of the figure. DAPI staining was used to visualize all the cells. (B) The percentages of GFP positive (GFP+) cells in basal layers were counted. *p<0.05. (C) Representative holoclone forming assay of HEKs and HLEKs following transduction of pCDH or miR-107 lentiviral constructs. miR-107 transduced keratinocytes gave rise to significantly more holoclones than the scrambled control. 100 cells per plate were seeded for each treatment. *p<0.05. (**D and E**) Luciferase assays to verify the interaction of Wnt3a with miR-103/107 using the psiCHECK^TM-2 vector harboring a wild (D) or mutant (E) 3’ untranslated region (UTR) of Wnt3a. Constructs were co-transfected with either miR-control, miR-103 or miR-107 into cells. N: precursor microRNA control, 103: precursor microRNA-103, 107: precursor microRNA-107. N.S.: Not Statistically Significant. (F) HLEKs were treated with either Ir-antago, Antago-103 or Antago-107 for 24 hours following concentrated culture media and lysates were immunoblotted for secreted wnt3a, SOX9 , phospho JNK and YAP1. (G) Immunoblotting was performed to check total Wnt3a expression following over-expression of either pre-miR-negative control (Nega), pre-miR-103, or pre-miR-107 in hTCEpi cells. Normalized expression to GAPDH is included. (H) Representative holoclone forming assay of HEKs following transduction of pCDH or miR-107 lentiviral constructs with or without Wnt3a (30ng/ml). Wnt3a inhibited enhanced holoclone formation induced by overexpression of miR-107. . 200 cells per plate were seeded for each treatment. *p<0.05. (I) Expression of SOX9 and p-JNK was determined in HLEKs following treatment of recombinant Wnt3a for 24 hours.

miR-103/107 family increases the proliferative capacity of keratinocytes by targeting Wnt3a

One can assess the proliferative capacity of epithelial cells via their ability to form holoclone colonies [49]. Colony-forming keratinocytes yield three distinct clonal types; holoclones, meroclones and paraclones [49]. Holoclone-forming keratinocytes have the greatest growth potential, give rise to the largest colonies and are considered to be “stem cells” [49]. If the miR-103/107 family increased the proliferative capacity of keratinocytes, ectopic expression of these miRNAs should yield more holoclone colonies. To address this question, HEKs and HLEKs were transduced with miR-107 or a scrambled control, were seeded at clonal density (100 cells/100 mm dish) [41] and allowed to grow for two weeks. Colonies were then fixed, stained and analyzed [41]. Indeed, the miR-107 transduced HEKs and HLEKs gave rise to significantly more holoclones (colonies >0.5cm diameter) than the scrambled control HEKs and HLEKs (Fig. 3C). Antago-107 treatment of HEKs markedly decreased holoclone colonies (Supporting Information S5). Such results demonstrate that the miR-103/107 family contributes to a stem cell phenotype by increasing the proliferative capacity of keratinocytes.

Low levels of Wnt3a are a characteristic of label-retaining cells (putative stem cells) isolated from the bulge region of the hair follicle [14, 50], the site of the hair follicle stem cells [8]. Interestingly, Wnt3a is a potential target of miRs-103-107, and this was demonstrated by luciferase assays (Figs. 3D and E). To further validate this observation, HLEKs and hTCEpi cells were treated with antagos-103/107 and levels of endogenous Wnt3a were determined. An increase in secreted Wnt3a was observed in HLEKs following antagomir treatment (Fig. 3F), whereas levels of secreted Wnt3a were lower following ectopic expression of miRs-103/107 (Fig. 3G), confirming our luciferase finding and establishing that in keratinocytes, Wnt3a is a target of miRs-103/107. We reasoned that if low levels of Wnt3a were involved in stem cell maintenance, exposure of miR-107-transduced keratinocytes to recombinant Wnt3a should impair the ability of these cells to form holoclone (stem cell-enriched) colonies. Wnt3a-treated keratinocytes had markedly less holoclone colonies compared with PBS-treated keratinocytes (Fig. 3H), and recombinant Wnt3a completely abrogated the miR-107-induced increase in holoclone colony formation (Fig. 3H). Thus by targeting Wnt3a, the miR-103/107 family are able to confer increased proliferative capacity to keratinocytes, which is a hallmark of stem cells.

With respect to hair follicle stem cells, it has been suggested that the canonical Wnt signaling pathway is irrelevant to stem cell maintenance [50]. Therefore, we concentrated our efforts on whether miRs103/107 targeting of Wnt3a involved the non-canonical signaling pathways. Reports indicate that JNK, which is a component of the non-canonical Disheveled (DVL-JNK pathway [51], can phosphorylate YAP1 [52]. Yap1 is a determinant of epidermal stem cell proliferative capacity [53, 54]. Interestingly, HLEKs treated with antagos-103/107 resulted in a marked increase in phospho-JNK (Fig. 3F) and a decrease in YAP-1 protein (Fig. 3F). Therefore, the up-regulation of p-JNK along with the inhibition of YAP1 by the loss of miRs-103/107 is consistent with a pro-stem cell role for these miRNAs. Another non-canonical pathway, the G protein-mediated Ca²⁺/CaMKII Wnt signaling pathway, ultimately results in the down regulation of Sox 9, which is a transcriptional target [55]. We observed that HLEKs treated with recombinant Wnt3a had lower Sox 9 and higher p-JNK levels compared with control treated HLEKs (Fig. 3I). Furthermore, upon exposure of HLEKs to antagos-103/107, Sox 9 levels decreased compared with irrelevant antagomir treatment (Fig. 3F). Our findings strongly suggest that miRs-103/107 targeting of Wnt3a results in the up-regulation of Sox 9 via the non-canonical Ca²⁺-mediated pathway and that the downregulation of p-JNK and YAP1 is also involved in the maintenance of keratinocyte self-renewal.

miR-103/107 family regulates E-cadherin

In order to self-renew, stem cells must remain within the niche [56]. E-cadherin (E-cad)-mediated cell adhesion appears to be a general mechanism for anchoring stem cells to their niches [56-58]. However, it is unclear how keratinocytes in stem cell niches maintain E-cad. Intriguingly, miRs-103/107 enhanced E-cad-based adherens junctions in HLEKs. Treatment of HLEKs with antagos-103/107 resulted in a loss of cell-cell contacts (Fig. 4A) and immuno-detectable E-cad at intercellular junctions after 6 h (Fig. 4B), when compared with irrelevant antago-treated (antago-124) HLEKs. A marked downregulation was noted in E-cad and p120 catenin levels (Fig. 4C), which are essential components of adherens junctions [59, 60]. We also determined E-cad expression in 3D-organotypic cultures (Supporting Information Fig. 6). In those cultures treated with antago-107, E-cad expression is markedly reduced.

In order to understand how miRs-103/107 function in maintaining limbal epithelial E-cad-mediated adhesion, we undertook a bioinformatics approach to determine candidate genes that had miR-103 and miR-107 binding sites within the 3’UTR. Of 19 predicated targets (Supporting Information Fig S7), only NEDD9, a scaffolding protein that negatively regulates localization and stability of E-cad [61]in a mammary epithelial cell line, was shown to be a direct target of miRs-103/107 in HeLa cells as well as in HLEKs and hTCEpi cells (Fig 4D and E). A significant reduction in NEDD9 protein was noted in hTECpi cells transduced with either pre-miR-103 or miR-107 compared with miR-negative controls (Fig 4F). Knocking down miRs-103/107 in HLEKs and hTCEpi cells with antagomiRs resulted in a significant increase in NEDD9 protein compared with an irrelevant antagomir (Fig. 4G). To determine whether NEDD9 promotes E-cad degradation via Src and FAK in keratinocytes, we determined changes in the levels of E-cad, Src, p-Src and p-FAK following antagomiR-treatment in HLEKs and hTCEpi cells (Fig. 4G). Along with an increase in NEDD9 protein, E-cad levels were decreased and p-Src (Y416) and p-FAK were increased (Fig. 4G). Silencing NEDD9 in HLEKs and hTCEpi cells with two different siRNA constructs decreased p-FAK (Y579/577 and Y925), as well as p-Src and p-p90RSK (Supporting Information Fig. S8). To confirm that NEDD9 induction is responsible for E-cad loss in HLEKs depleted of miRs-103/107, we conducted a rescue experiment using siNEDD9 in conjunction with antagos-103/107 (Fig. 4H). As expected, antagomir treatment reduced E-cad levels whereas these levels were dramatically increased in NEDD9-deficient HLEKs following antagos-103/107 treatment. In addition, we overexpressed Nedd9 using a construct without the miRs-103/107 binding site and such overexpression rescued the miR-103/107-induced increase in E-cad (Supporting Information Fig. S9). Collectively, these rescue experiments are strong evidence for the regulation of E-cad via a miR-103/107/NEDD9 axis.

miR-103/107 family regulates gap junctions

Interestingly, knocking down miRs-103/107 in HLEKs and hTCEpi cells with antagomiRs resulted in an increase in the gap junction protein Cx43 (Fig. 5A), as well as localization of this protein to cell-cell junctions (Fig. 5C). Consistent with the increase in Cx43, was the numerous gap junctions present between adjacent HLEKs and hTCEpi cells (Fig. 5B). hTCEpi cells treated with antogos-103/107 and scrape loaded to introduce Luciferase yellow dye, showed increased transmission of the dye into adjacent cells compared with control hTCEpi cells (ir-antagomir) (Fig. 5C) indicative of communication competency.

Some tyrosine phosphatases positively regulate gap junctions [62]. There are seven tyrosine phosphatases that are predicted targets of miRs-103/107 (Supporting Information Fig. S10). We demonstrated the direct interaction between miR-103/107 and its novel target, PTPRM by luciferase assays (Figs. 5D and E; Supporting Information Fig. S10) and western blotting (Figs. 5F-H and J). A significant reduction in both the precursor and the membrane-bound (p-subunit) portion of PTPRM proteins were noted in hTCEpi cells transduced with either pre-miR-103 or pre-miR-107 compared with miR-negative controls (Fig. 5F). Antagos-103/107 treatment resulted in increased levels of the p-subunit of PTPRM in HLEKs and hTCEpi cells (Fig. 5G). The PΔE (lack of extracellular portion) form was also increased by antago-103/107 treatment (data not shown). Similarly, silencing PTPRM with two different siRNAs decreased Cx43 protein levels in HLEKs and hTCEpi cells (Fig. 5H). This is consistent with the overexpression of miRs-103/107 in hTCEpi cells which resulted in a decrease in Cx43 protein (Fig. 5F). Cx43 was readily detected in hTCEpi cells lacking miRs-103/107 (antago-treated) (Figs. 5J and I). Cx43 protein levels could be reversed in such antago-103/107-treated cells with the inhibition of PTPRM using either a broad-spectrum phosphatase inhibitor or siRNA pool against this PTP (Figs. 5J and I). Collectively, these findings demonstrate that PTPRM regulates Cx43 levels in keratinocytes and supports the idea that gap junctions are regulated, in part, through a miR-103/107/PTPRM interaction.

Limbal epithelial processes regulated by the miR-103/107 family

Since miRNAs are known to regulate a plethora of targets [63], we evaluated the spectrum of targets affected by miRs-103/107. We knocked down miR-103 and miR-107 in primary cultures of human limbal epithelial keratinocytes using an antagomir approach (Supporting Information Figs. S4 A-C) and used mRNA profiling combined with bioinformatic analysis to identify miRNA targets in an unbiased approach [64] and evaluated the processes regulated by this miRNA family in these cells (Supporting Information Fig. S12). Not surprisingly, cell proliferation and cell-cell contact were two of the main functions of miRs-103/107 in HLEKs (Supporting Information Fig. S12). Bioinformatic analysis suggested seventeen additional putative targets regulated by the miR-103/107 family (Supporting Information Table 3). To validate the expression profiling data, we chose four potential targets (SLC2A3, DUSP5, CREB5, MID1) and confirmed their up-regulation by antagos-103/107 using qPCR analysis (Supporting Information Fig. S13). The expression profiling analyses strengthen our finding that the miR-103/107 family provides the means to regulate proliferation and cell-cell communication in keratinocytes from the stem cell-enriched limbal epithelium.

Discussion

Our study is the first to implicate miRs-103/107 as a cell autonomous factor that arrests cells in the G0/G1 phase of the cell cycle and enhances proliferative capacity; features associated with stem cells. Mature miR-103 and miR-107 have identical sequences except for one nucleotide at the 3’-end, and regulate overlapping targets (Trajkovski et al., 2011; Chen et al., 2012;Zhang et al, 2012). Similarly, we have observed that NEDD9, Wnt3a, PTPRM and p90RSK2 are novel targets for both miR-103 and miR-107.

miRs-103/107 as an Important Regulator of Cell-Cell Communication

By targeting NEDD9, miRs-103/107 maintain E-cadherin levels in HLEKs and HEKs. NEDD9, also known as human enhancer of filamentous 1 (HEF1) is a multidomain scaffolding protein of the Cas family [65]. In keratinocytes, NEDD9 negatively regulates E-cadherin and promotes E-cadherin degradation via Src and FAK since attenuating miRs-103/107 levels decreased E-cad and increased NEDD9 along with Src and Fak (Fig. 4G). Cadherins are transmembrane proteins that are components of adherens junctions (AJs) and promote cell adhesion, especially E-cadherin. Limbal niche cells, which are a population of limbal basal epithelial cells [13], have high levels of E-cad [66]. Our data unequivocally demonstrates that miRs-103/107 positively regulates E-cad and this provides an explanation for the high expression of E-cad in the limbal niche cells. We posit that such an overabundance of miRs-103/107 in the limbal epithelium may reflect the need to ensure the integrity of the limbal epithelial stem cell niche. In this context, E-cad plays a major role in maintaining stem cells [67]. In many systems such as Drosophila gonads [68, 69] and human and mouse ESC and iPSC [70-72], E-cad expression ensures the retention of non-differentiated cells in the niche by rapid displacement of the differentiated cells and mediates the self-renewal potential of SCs [68, 69]. It is well accepted that limbal epithelial basal cells are biochemically less differentiated than the corneal epithelial basal cells [4, 73, 74] and E-cad might help maintain these cells in the limbal niche. Conversely, E-cad also has a pro-differentiation role in keratinocytes [75], which is consistent with its strong expression in suprabasal layers of stratified squamous epithelia. In this regard, both limbal and corneal epithelia express E-cadherin [76, 77], thus the limbal-preferred expression of miRs-103/107 indicates that E-cad is, in part, regulated differently in these two regions of the ocular anterior segment.

Our observation that miRs-103/107 target PTPRM and thus attenuate Cx43 expression in epidermal and limbal epithelial keratinocytes provides a mechanism for the finding that this protein is poorly expressed in the stem cell-enriched regions of epidermis [78], limbal epithelium [79, 80 ] and hair follicle [78]. Since Cx43 is associated with a more differentiated cell phenotype [78, 80], its absence in these stem cell-enriched basal cell populations is not surprising. In the limbal epithelium, lack of gap junction-mediated cell-cell communication was hypothesized to be a mechanism by which limbal stem cells maintained stemness [81]. Interestingly, in neural progenitor cells, inhibition of gap junctions induced a slow cycling phenotype, characterized by slow progression, but not exit, through the cell cycle [82]. These authors hypothesized that the circadian clock gene per2, which arrests cells in the cell cycle [83] and is sensitive to intracellular calcium [82], could be influenced by gap junction-dependent, calcium transport. In other systems, gap junction communication has been associated with induction of stem cell differentiation. For example, Cx43 negatively regulates stem cell self-renewal as well as cancer stem cell renewal [84, 85] and the introduction of miR-499 into cardiac stem cells via gap junctions induced stem cell differentiation [86].

miRs-103/107 Coordinate Proliferation with Cell-Cell Communication

It is interesting that a single miRNA family negatively regulates Cx43 as well as p90RSK2; both of which are associated with quiescence. This suggests that miRs-103/107 coordinate cell-cell communication with proliferation in a cell autonomous manner. One of the hallmarks of adult epithelial stem cells is their normally quiescent state [3, 6, 8], where the stem cell progeny (TA cells) cycle relatively frequently and provide the cells necessary to maintain the normal steady-state conditions of self-renewing epithelia [87]. Directly related to regulation of the quiescent status of epithelial stem cells is the finding that C/EBPδ controls mitotic quiescence of limbal keratinocytes by holding cells in the G₀/G₁ phase of the cell cycle [88]. Ectopic expression of C/EBPδ increases keratinocyte cell cycle length via activation of p27^Kip1 [88]. Furthermore, it was postulated that co-expression of Bmi1 and C/EBPδ in limbal keratinocytes conferred both quiescence and proliferative capacity [88] In the present study, we demonstrate that miRs-103/107 arrest limbal keratinocytes in the G0/G1 phase of the cell cycle by targeting p90RSK2, which is an isoform of the p90 ribosomal S6 kinase [89]. Interestingly, this kinase has not previously been associated with keratinocyte proliferation; however it regulates G1 progression in human embryonic kidney 293T cells by phosphorylating p27^Kip1 [90] in a manner similar to C/EBPδ. To date, there is no indication that these pathways overlap, indicative that more than one post-transcriptional mechanism regulates p27^Kip1 in keratinocytes. In support of the idea that attenuation of p90RSK2 by miRs-103/107 regulates slow-cycling in the stem cell-enriched limbal epithelium, cancer stem cells from MCF7 cells also express low levels of p90RSK [91],

miR-103/107 Family Enhances Proliferative Capacity via Wnt3a Repression

Ectopic expression of miR-107 in primary human epidermal and limbal cultures increased the potential of these keratinocytes to form holoclone colonies, a measure of proliferative capacity and an accepted in vitro marker of stem cells [49]. This implies that this miRNA family confers a degree of “stemness” to these cells. That miRs-103/107 increase proliferative capacity and arrest cells in the G0/G1 phase of the cell cycle might appear paradoxical. However, epithelial stem cells are normally quiescent but upon stimulation are capable of undergoing numerous rounds of division [6, 87, 92]. That a single miRNA family can regulate such divergent processes extends and strengthens the idea that proliferation and self-renewal potential are two related but discrete processes.

The mechanism underlying miRs-103/107 enhancement of proliferative capacity involves, in part, the Wnt signaling pathway. However, Wnt-β-catenin signaling is not a requirement for proliferative capacity of hair follicle stem cells but is necessary for stem cell fate determination [50]. Our findings implicate attenuation of the non-canonical JNK-DVL and Ca⁺²-CMKII pathways, which enhances limbal and epidermal keratinocyte proliferative capacity Endogenous Wnt activity is normally low in the unperturbed limbal epithelium [93-95], Similarly, Wnt 3A was minimally detected in limbal epithelial precursor and limbal niche cells [93] as well as in the stem cell-enriched bulge region of the telogen hair follicle [14, 50]. On the other hand, activation of Wnt signaling in limbal cells resulted in an increase in proliferation and colony forming efficiency [94]; however, this result was obtained by treating limbal keratinocytes with lithium chloride, which has a myriad of different cellular and biochemical effects that are both dependent and independent of GSK3β inhibition [96]. Taken together, most in vivo findings implicate a role for non-canonical Wnt signaling in stem cell maintenance.

In light of the present work, the Ca²⁺-mediated and DVL/JNK non-canonical Wnt signaling pathways may be responsible for maintaining keratinocyte stemness. One of the effects of the Ca²⁺-mediated pathway is repression of Sox 9 [55]. Recent studies have revealed that in hair follicle stem cells, loss of Sox 9 results in their differentiation into epidermal cells and eventual stem cell depletion [97]. Our data indicates that miRs-103/107 positively regulate Sox 9 via Wnt 3A repression which may contribute to increased numbers of holoclone colonies. This is consistent with Sox 9 being a positive regulator of stem cells [97]. YAP1, a co-activator of the Hippo pathway and a regulator of the cancer cell phenotype [98-100] can directly up-regulate Sox 9 [101] and be phosphorylated by JNK[52]. YAP1 is a determinant of epidermal stem cell proliferative capacity [53, 54] and our data suggests that a Wnt3a/YAP1/Sox 9 nexus may underlie how miRs-103/107 increase the ability of limbal and epidermal keratinocytes to form holoclone colonies. In support of this idea, loss of miRs-103/107 result in a marked increase in p-JNK levels in HLEKs and HEKs. JNK specifically phosphorylates endogenous YAP1 in several cell types including HaCaT keratinocytes [52]. Such phosphorylation results in cytoplasmic retention of YAP and its repression [98]. Thus we postulate that in keratinocytes, miRs-103/107 targeting of Wnt3a through the non-canonical DVL/JNK signaling pathway, bypasses JNK-mediated YAP repression, which enables a YAP 1/Sox 9 co-mediated regulation of proliferative capacity. It is interesting that both the Ca²⁺-mediated and DVL/JNK non-canonical Wnt signaling pathways are involved in enhancing keratinocyte proliferative potential.

miRs-103/107 in ex vivo stem cell expansion

One of the major challenges of ex vivo epidermal and corneal epithelial cell therapy is expansion and preservation of “stemness” from a small biopsy. Initially, expansion of limbal epithelial cells was accomplished on a 3T3 fibroblast feeder layer [36, 102]. More recently human amniotic membrane has been used as a platform for the expansion of limbal keratinocytes based on the idea that this membrane recapitulates critical aspects of the limbal epithelial stem cell niche (see [103] and references therein). Since ex vivo expansion of limbal-derived stem cells is a crucial step in treating corneal blindness, we believe that ectopic expression of miRs-103/107 in conjunction with the newly optimized amniotic membrane protocols may represent a novel means of expanding the proliferative capacity of the limbal-derived keratinocytes. Since human epidermal keratinocytes behave similarly as limbal keratinocytes with respect to proliferative capacity, ectopic expression of miRs-103/107 may effectively increase epidermal keratinocytes needed for regeneration.

Supplementary Material

Supp FigureS1-S13

Figure S1. (A) microRNA qPCR analysis of mature miR-103 and miR-107 levels in corneal and limbal epithelia. Values are means ± SD of three independent experiments. *p <0.05. (B) In situ hybridization with a digoxygenin-labeled antisense probes for miR-107 and miR-103 of human corneal and limbal epithelia as well as mouse skin.

Figure S2. (A) microRNA qPCR analysis of miR-103 and miR-107 levels in miRs-103/107 lentivirally transduced cells. Values are means ± SD of three independent experiments. (B) Population doubling times were measured in cal27 cells transduced with either miR-103, miR-107 or empty vector (pCDH). Values are means ± SD of three independent experiments.

Figure S3. Doubling time of hTCEpi cells following transfection of either siRNA control or siRNA against RSK2.

Figure S4. (A and B) Mature miR-103/107 levels were determined by TaqMan microRNA assay after treatment of Ir-antago, antago-103 or miR-107 in HLEKs and hTCEpi cells. Relative ratios of mean value are indicated as fold-change compared to Ir-antago, which is assigned a value of “1”. (C) Antago-103 or 107 had no effect on the expression of another randomly chosen mature miRs-31, 184, 203 and 205 indicating the specificity of antagomirs for miR-103/107. Expression levels were calculated relative to 18S rRNA. Relative gene expression was calculated using the comparative C_T method. N.S.: Not Statistically Significant.

Figure S5. Representative holoclone forming assay of HEKs following two-day antagomir treatments. Antago-107 treated keratinocytes gave rise to significantly less holoclones than the Ir-antago treated cells. 200 cells per plate were seeded for each treatment. *p<0.05.

Figure S6. E-cadherin immunostaining of 12 d 3D organotypic raft cultures derived from HEKs. Antagomirs were treated at day 3 for 3 days. Antago-107 treatment significantly decreased E-cad levels compared with Ir-antago treated rafts. Images taken with a 20x objective.

Figure S7. Screening of predicted target genes of miR-103/107 using the psiCHECK^TM-2 constructs harboring a 3’UTR of each gene. Predicted target genes were selected based on negative effects on E-cadherin. The construct was co-transfected with either precursor miR control or precursor miR-107 into HeLa cells. Twenty four hour after transfection, cell lysates were used to measure both firefly and renilla luciferase activities using the Dual-Luciferase® Reporter Assay System. N: precursor microRNA control, 107: precursor microRNA-107. N.S.: Not Statistically Significant.

Figure S8. Immunoblotting of total NEDD9, phospho-Src, phospho-FAK and phospho-p90RSK (S380) in hTCEpi cells transfected with either control siRNA (siCon) or siNEDD9.

Figure S9. Immunoblotting of Nedd9, E-cad and GAPDH in hTCEpi cells. miRs-103/107 mimics were transfected into hTCEpi cells with or without overexpression of Nedd9.

Figure S10. Screening of predicted target genes of miR-103/107 using the psiCHECK^TM-2 constructs harboring a 3’UTR of each gene. The construct was co-transfected with either precursor miR control or precursor miR-107 into HeLa cells. Twenty four hour after transfection, cell lysates were used to measure both firefly and renilla luciferase activities using the Dual-Luciferase® Reporter Assay System. N: precursor microRNA control, 107: precursor microRNA-107. N.S.: Not Statistically Significant.

Figure S11. Schematic diagrams of miR-103/107 binding sites in the 3’UTR region of RSK2, Wnt3a, NEDD9 and PTPRM mRNAs. Mutant reporter constructs were generated at the first three nucleotides of the seed-match sequence (bold red).

Figure S12. (A) Gene Ontology (GO) terms for differential expressed genes in antago-103/107 treated HLEKs when compared with Ir-antago treated HLEKs. (B) Venn diagram showing the common and unique targets by miR-103 and miR-107. It demonstrated that seventeen predicted target genes were targeted by both miRs-103/107.

Figure S13. Real time qPCR analysis of SLC2A3, DUSP5, CREB5 and MID1 levels in HLEKs that were treated with an Ir-antago, Antago-103, or Antago-107 for 6 h. Values are means ± SD of four independent experiments.

NIHMS658893-supplement-Supp_FigureS1-S13.pdf^{(2.8MB, pdf)}

Supp TableS1-S4

NIHMS658893-supplement-Supp_TableS1-S4.docx^{(24.9KB, docx)}

Acknowledgements

Primary epidermal keratinocyte cultures and 3-D organotypic raft cultures were obtained from the Northwestern University Skin Disease Research Center (NU-SDRC) Skin Tissue Engineering Core facility; lentiviral constructs were obtained from the NU-SDRC DNA/RNA Delivery Core facility; and the NU-SDRC Morphology and Phenotyping Core facility assisted in morphological analysis. The NU Genomic Core assisted in the mRNA expression profiling studies.

The NU-SDRC is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR057216. This research is supported by National Institutes of Health Grants EY06769, EY017536 and EY019463 (to R.M.L.) and AR062110 (to S.G.); a Dermatology Foundation research grant and Career Development Award (to H.P.); and a MidWest Eye-Banks research grant (H.P.).

Footnotes

Conflict of interest

The authors declare that they have no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigureS1-S13

Figure S3. Doubling time of hTCEpi cells following transfection of either siRNA control or siRNA against RSK2.

Figure S8. Immunoblotting of total NEDD9, phospho-Src, phospho-FAK and phospho-p90RSK (S380) in hTCEpi cells transfected with either control siRNA (siCon) or siNEDD9.

Figure S9. Immunoblotting of Nedd9, E-cad and GAPDH in hTCEpi cells. miRs-103/107 mimics were transfected into hTCEpi cells with or without overexpression of Nedd9.

NIHMS658893-supplement-Supp_FigureS1-S13.pdf^{(2.8MB, pdf)}

Supp TableS1-S4

NIHMS658893-supplement-Supp_TableS1-S4.docx^{(24.9KB, docx)}

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PERMALINK

microRNA-103/107 family regulates multiple epithelial stem cell characteristics

Han Peng

Jong Kook Park

Julia Katsnelson

Nihal Kaplan

Wending Yang

Spiro Getsios

Robert M Lavker

Abstract

Introduction

Material and Methods

Mice

Cell culture, transduction and transfection

Antagomirs

Laser capture microdissection and microRNA profiling

mRNA expression profiling

In situ hybridization

Cell cycle analysis

Western Blotting

Immunofluorescence and Immunocytochemistry

Colony Formation assay

Progenitor competition assay

Real time qPCR

Inhibitors

Scrape loading dye transfer assay

Luciferase Reporter Constructs and Assay

Statistical analysis

Results

miR-103/107 family is limbal epithelial-preferred

miR-103/107 family arrests keratinocytes in G0/G1

Figure 1.

miR-103/107 family targets p90RSK2

Figure 2.

miR-103/107 family maintains keratinocytes in the basal layer

Figure 3.

miR-103/107 family increases the proliferative capacity of keratinocytes by targeting Wnt3a

miR-103/107 family regulates E-cadherin

Figure 4.

miR-103/107 family regulates gap junctions

Figure 5.

Limbal epithelial processes regulated by the miR-103/107 family

Discussion

miRs-103/107 as an Important Regulator of Cell-Cell Communication

miRs-103/107 Coordinate Proliferation with Cell-Cell Communication

miR-103/107 Family Enhances Proliferative Capacity via Wnt3a Repression

miRs-103/107 in ex vivo stem cell expansion

Supplementary Material

Acknowledgements

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

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