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MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

Mun J, Tam C, Chan G, Kim JH, Evans D, Fleiszig S - PLoS ONE (2013)

Bottom Line: Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization.Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir.Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, University of California, Berkeley, California, United States of America.

ABSTRACT
Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

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Tear fluid induction of miR-762 negatively regulates RNase7 and ST2 gene expression.a Tear fluid induction of miR-762 expression in corneal epithelial cells was reduced by the antagomir of miR-762. b The antagomir of miR-762 enhanced tear-induced expression of RNase7 and ST2 mRNA compared to control (scrambled) antagomir suggesting that tear-induced miR-762 expression negatively regulates these innate defense genes. RT-PCR was used to measure gene expression at 48 h after transfection (see Methods). (* p < 0.05, t-Test versus tear-treated scrambled controls).
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pone-0057850-g004: Tear fluid induction of miR-762 negatively regulates RNase7 and ST2 gene expression.a Tear fluid induction of miR-762 expression in corneal epithelial cells was reduced by the antagomir of miR-762. b The antagomir of miR-762 enhanced tear-induced expression of RNase7 and ST2 mRNA compared to control (scrambled) antagomir suggesting that tear-induced miR-762 expression negatively regulates these innate defense genes. RT-PCR was used to measure gene expression at 48 h after transfection (see Methods). (* p < 0.05, t-Test versus tear-treated scrambled controls).

Mentions: Having shown tear-antigen upregulation of miR-762, and miR-762 negative regulation of genes encoding RNase7 and ST2 without tears, we next used a miR-762 antagomir to test the relationship between tear exposure, miR-762 induction, and RNase7 and ST2 mRNA expression. Corneal epithelial cells were transfected with Antago-762 or an irrelevant antagomir (control), then exposed to cell culture media or tear fluid for 16 h and tested for the expression of endogenous miR-762. Tear fluid alone upregulated the expression of miR-762 (∼ 4-fold), which was partially reduced by the miR-762 antagomir (Fig. 4 a). The reduction in tear-induced miR-762 upregulation in antagomir treated cells versus scrambled controls was significant (p < 0.05, t-Test). In the presence of antagomir, tear fluid induced an even greater expression of RNase7 or ST2 mRNA than that found in the presence of scrambled controls (Fig. 4 b, p < 0.05, t-Test, for each comparison) confirming that miR-762 serves to negatively regulate the expression of these tear-induced innate defense genes.


MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

Mun J, Tam C, Chan G, Kim JH, Evans D, Fleiszig S - PLoS ONE (2013)

Tear fluid induction of miR-762 negatively regulates RNase7 and ST2 gene expression.a Tear fluid induction of miR-762 expression in corneal epithelial cells was reduced by the antagomir of miR-762. b The antagomir of miR-762 enhanced tear-induced expression of RNase7 and ST2 mRNA compared to control (scrambled) antagomir suggesting that tear-induced miR-762 expression negatively regulates these innate defense genes. RT-PCR was used to measure gene expression at 48 h after transfection (see Methods). (* p < 0.05, t-Test versus tear-treated scrambled controls).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585208&req=5

pone-0057850-g004: Tear fluid induction of miR-762 negatively regulates RNase7 and ST2 gene expression.a Tear fluid induction of miR-762 expression in corneal epithelial cells was reduced by the antagomir of miR-762. b The antagomir of miR-762 enhanced tear-induced expression of RNase7 and ST2 mRNA compared to control (scrambled) antagomir suggesting that tear-induced miR-762 expression negatively regulates these innate defense genes. RT-PCR was used to measure gene expression at 48 h after transfection (see Methods). (* p < 0.05, t-Test versus tear-treated scrambled controls).
Mentions: Having shown tear-antigen upregulation of miR-762, and miR-762 negative regulation of genes encoding RNase7 and ST2 without tears, we next used a miR-762 antagomir to test the relationship between tear exposure, miR-762 induction, and RNase7 and ST2 mRNA expression. Corneal epithelial cells were transfected with Antago-762 or an irrelevant antagomir (control), then exposed to cell culture media or tear fluid for 16 h and tested for the expression of endogenous miR-762. Tear fluid alone upregulated the expression of miR-762 (∼ 4-fold), which was partially reduced by the miR-762 antagomir (Fig. 4 a). The reduction in tear-induced miR-762 upregulation in antagomir treated cells versus scrambled controls was significant (p < 0.05, t-Test). In the presence of antagomir, tear fluid induced an even greater expression of RNase7 or ST2 mRNA than that found in the presence of scrambled controls (Fig. 4 b, p < 0.05, t-Test, for each comparison) confirming that miR-762 serves to negatively regulate the expression of these tear-induced innate defense genes.

Bottom Line: Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization.Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir.Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, University of California, Berkeley, California, United States of America.

ABSTRACT
Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

Show MeSH
Related in: MedlinePlus