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Gary Hin-Fai Yam, Yufei Teng, Dennis Shun-Chiu Lam, Chi-Pui Pang; MicroRNA-145 Regulates Human Corneal Epithelial Differentiation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):295.
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We have identified the unique expression of hsa-miR-145 in human limbal parabasal epithelium. This study was to investigate the role of miR-145 in human corneal epithelial differentiation.
Human limbal-peripheral corneal epithelium was dissociated for primary corneal epithelial progenitor cell (CEPC) culture in CnT-20 medium (CellnTec). At early passage, cells showing holoclone formation ability were transfected with pMIRH145PA-1 or control pCDH-CMV-MCS-EF1 vector (System Biosciences). They were then tested for corneal marker expression by immunofluorescence and for the epithelium-forming ability by organotypic culture. Global gene expression was examined using Agilent Whole Human Genome Oligo Microarray platform, followed by Gene Ontology using GeneSpring GX version 11.0. Post-transcriptional regulation of miR-145 was determined by 3’UTR-luciferase reporter assay.
Human CEPCs transfected with miR-145 had higher expression of cytokeratin-3/12 and connexin-43 (corneal differentiation markers) and gave rise to defective epithelium in organotypic culture, when compared to cells with scrambled sequences. By oligo microarray analysis and with at least 5-fold difference compared to cells with scrambled sequences, miR-145-transfectedc cells had 324 up-regulated genes and 277 down-regulated genes. Significant pathway analysis highlighted the gene effect on immune and infection responses, cell proliferation, epithelial development and stem cell maintenance. Validated by qPCR, miR-145 up-regulated interferon beta-1 (P<0.005, paired Student’s t test) and down-regulated integrin beta-8 (ITGB8, P=0.00024). The direct binding to ITGB8 3’UTR was confirmed when miR-145 suppressed the expression of luciferase reporter cis-regulated by ITGB8 3’UTR.
Our results demonstrated the importance of miR-145 in corneal epithelial differentiation and formation. It could be involved in the maintenance of epithelial integrity and immune protection of the cornea.
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