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Male C57 mice received 10 consecutive daily intraperitoneal injections of melatonin, 5-methoxytryptamine or 5-methoxytryptophol (5mg/kg body weight). Control mice received the alcoholic saline vehicle. All mice were sacrificed 24 hours after the last injection. Following extraction of RNA from peritoneal exudate cells (PEC) and splenocytes, the level of gene expression was analyzed with the reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that melatonin up-regulated the level of gene expression of transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF-alpha) and stem cell factor (SCF) in PEC, and the level of gene expression of interleukin-1beta (IL-1beta), M-CSF, TNF-alpha, interferon-gamma (IFN-gamma) and SCF in splenocytes. 5-Methoxytryptamine augmented the level of gene expression of TGF-beta, M-CSF and SCF in PEC, and the level of gene expression of IL-1beta, TNF-alpha, IFN-gamma, M-CSF and SCF in splenocytes. 5-Methoxytryptophol elevated the level of gene expression of TNF-alpha, IL-1beta, TGF-beta and M-CSF in PEC, and the level of gene expression of inducible nitric oxide synthase (iNOS), IL-1beta, M-CSF, TNF-alpha, IFN-gamma and SCF in splenocytes.
BackgroundEpigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Methodology/Principal FindingsHuman LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.Conclusion/SignificanceWe found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.
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