Abstract
MicroRNAs are critical regulators of stem cell behavior. The miR-103/107 family is preferentially expressed in the stem cell-enriched corneal limbal epithelium and plays an important role in coordinating several intrinsic characteristics of limbal epithelial stem cells. To elucidate further the mechanisms by which miRs-103/107 function in regulating limbal epithelial stem cells, we investigate the global effects of miRs-103/107 on gene expression in an unbiased manner. Using antagomirs-103/107, we knocked down endogenous miRs-103/107 in keratinocytes and conducted an mRNA profiling study. We show that miRs-103/107 target mitogen-activated protein kinase kinase kinase 7 (MAP3K7) and thereby negatively regulate the p38/AP-1 pathway, which directs epithelial cells towards a differentiated state. Pharmacological inhibition of p38 increases holoclone colony formation, a measure of proliferative capacity. This suggests that the negative regulation of p38 by miRs-103/107 contributes to enhanced proliferative capacity, which is a hallmark of stem cells. Since miRs-103/107 also promote increased holoclone colony formation by regulating JNK activation through non-canonical Wnt signaling, we believe that this microRNA family preserves “stemness” by mediating the crosstalk between the Wnt/JNK and MAP3K7/p38/AP-1 pathways.
Highlights
Cornea is comprised of three layers: epithelium, stroma, and endothelium
Among hundreds of microRNAs, we identified nine that were highly expressed in the stem cell-enriched limbal epithelial basal cells compared with TA cellenriched central corneal epithelial basal cells [28]
For example (i) day 3 –K14 appears primarily in the basal cells [33, 34]; (ii) day 7 –there is a marked increase in corneal epithelial proliferation [35]; (iii) day 14 –is time of eye opening; and; (iv) day 60 –reflects a mature limbal epithelium [36]
Summary
All the procedures involving animals were approved by the Northwestern University Animal Care and Use Committee (NUACUC). A PALM laser capture system (LCM; Zeiss) was used to isolate basal cells from limbal epithelium as previously described [28,29,30]. Functional Annotation Clustering of known target genes by specific microRNAs was performed in DAVID Functional Annotation Bioinformatics Resources v6.7. HLEKs were treated with antagomir-103,-107 and irrelevant-antagomir and subsequently total cellular RNAs were isolated and purified by an mRNeasy kit (Qiagen, Hilden, Germany). Seed nodes included: (i) the differentially expressed genes in antagomir-103/107-treated HLEKs (S2 and S3 Tables) and (ii) stem-cell-maintenance-related direct target genes of miRs-103/107 (S4 Table). As previously described [28], HLEKs (200 cells per 100 mm dish) were seeded and incubated in FAD medium (DMEM/F12 media, FCS (10%), insulin (5 μg/ml), adenine (0.18 mM), hydrocortisone (0.4μg/ml), cholera toxin (10ng/ml), triiodothyronine(5μg/ml), 5μg/ml Human apotransferrin, glutamine (4mM), penicillin-streptomycin (50 IU/ml), and Epidermal growth factor (10 ng/ml)) with mitomycin C-treated 3T3 feeder cells. The significance of the differences between 3 groups was tested by non-parametric oneway ANOVA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
