Abstract
Purpose: :
To identify the corneal progenitor cell-specific microRNAs and elucidate their roles in cell proliferation, apoptosis and progenitor cell maintenance.
Methods: :
Fresh human corneal rims leftover from corneal transplantation were recruited from Joint Shantou International Eye Centre, Shantou, China. MicroRNA was enriched from limbal epithelium and central corneal epithelium by Trizol method. MicroRNA was profiled using Human microRNA Microarray Kit (V2) (Agilent Technologies) which screens for expression of 723 human microRNAs from the Sanger database v.10.1. Results were validated by quantitative polymerase chain reaction (qPCR). Localization of target microRNAs in frozen corneal tissue sections were examined by in situ hybridization using locked nucleic acid (LNA)-modified oligonucleotide probes. The biological role of target microRNAs in human corneal epithelial cell line (HCE), human and mouse primary corneal progenitor cells was investigated by over-expression experiments using lipophilic transfection of precursor microRNAs. Characterization of transfected cells was performed with cell growth, cell cycle changes and expression of stem cell and differentiation markers.
Results: :
Four independent microRNA microarray analyses showed that hsa-miR-143 and hsa-miR-145 were significantly up-regulated in limbal epithelium enriched with corneal progenitor cells when comparing to central cornea epithelium depleted of progenitor cells (P<0.05, paired Student’s t-test). Validation by qPCR on eleven human corneal samples identified a ΔCT of 5.9±0.8 in limbal epithelia and 11.1± 0.9 in more differentiated central corneal epithelia (P=0.0006, Mann Whitney U-test) for hsa-miR-143 expression, and a ΔCT of 4.5±0.7 in limbal epithelia and 10.2±0.7 in central corneal epithelia (P=0.0004, Mann Whitney U-test) for hsa-miR-145. Transfection with pre-miR-145 to human corneal epithelial cells resulted in more than 3-fold decrease of proliferation by MTT assay (compared to scramble control). The cells were shifted to G2/M arrest by flow cytometry and there was formation of holoclone-like colonies.
Conclusions: :
We identified hsa-miR-143 and 145 cluster specific to corneal progenitor cells resided in the limbus epithelium. MiR-145 showed a possible role in corneal progenitor cell regulation and proliferation. Our work was the first to elucidate the role of microRNAs on corneal cell homeostasis.
Keywords: cornea: epithelium