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James J. Mun, Connie Tam, David J. Evans, Suzanne M. Fleiszig; Expression Of MicroRNAs In Human Corneal Epithelial Cells Is Modified By Exposure To Human Tear Fluid And Pseudomonas Aeruginosa Antigens. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1942.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown that human tear fluid acts directly upon corneal epithelial cells to reduce their susceptibility to multiple virulence strategies of P. aeruginosa. This phenomenon is accompanied by changes to the expression of a large number of genes including some known to be potent antimicrobials at other epithelial sites, and others known to provide protection against bacterial keratitis. MicroRNAs are endogenous, non-coding, regulatory RNAs that inhibit the translation of target mRNA. MiRNAs are predicted to be involved in most biological processes through negative and positive regulation. Here we examined miRNA expression in corneal epithelial cells response to tear fluid with and without exposure to bacterial antigens to advance our understanding of how tear fluid modulates epithelial cell responses to bacteria (and corneal gene expression).
Human corneal epithelial cells were incubated with human tear fluid or cell culture media for 6 h, or to tear fluid or media for 16 h followed by 3 h incubation with P. aeruginosa antigens. Total RNA was isolated from the epithelial cells, first strand cDNA synthesized and used to generate cRNA, which was fragmented and applied to Affymetrix GeneChip miRNA Arrays. MiRNA QC Tool software was used for analysis and genes up- or downregulated by at least 2-fold compared to controls were chosen for further study.
Tear fluid alone (6 h) upregulated 8 microRNAs (e.g., miR-1228, miR-23a) and downregulated 5 microRNAs (e.g., miR-188, miR-205) in corneal epithelial cells by 2-fold or more. Pre-exposure to tear fluid alone (16 h) followed by exposure to P. aeruginosa antigens upregulated 6 microRNAs (e.g., miR-1207, miR-762, let-7c) and downregulated 12 microRNAs (e.g., miR-126, miR-205, let-7b, let-7e).
Tear fluid modulated expression of multiple miRNAs in corneal epithelial cells. Down regulation of miR-205 in both treatment groups is of interest considering that suppression of miR-205 dampens Akt signalling via SHIP2 induction in corneal epithelial cells, and that Akt signalling is involved in intracellular routing of P. aeruginosa. It is yet to be determined if miR-205 and/or other miRNAs differentially expressed by tear fluid exposure, relate to mechanisms by which tear fluid modulates cell defenses against bacteria (or other effects of tear fluid on cell biology).
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