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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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The effect of a miR-762 antagomir on gene expression in human corneal epithelial cells and on P. aeruginosa invasion.a Transfection of human corneal epithelial cells with an antagomir of miR-762 (antago-762) decreased the expression of miR-762 relative to a control (scrambled) antagomir, and b increased the expression of Rab5a mRNA, a predicted target of miR-762. c Antago-762 also increased the expression of RNase7 and ST-2 mRNA suggesting that miR-762 negatively regulates these innate defense genes. Antago-762 did not affect genes encoding hBD-2 and hBD-3. d The antagomir did not affect epithelial susceptibility to P. aeruginosa invasion. Gene expression was measured by real-time PCR, and P. aeruginosa invasion by using gentamicin exclusion assays. In each experiment, cells were used at 48 h after transfection. For invasion assays, epithelia were challenged at 72 h with 104 cfu invasive P. aeruginosa strain 6294 for 3 h followed by 1 h gentamicin treatment (see Methods). (* Significant difference vs. respective controls, t-Test).
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pone-0057850-g002: The effect of a miR-762 antagomir on gene expression in human corneal epithelial cells and on P. aeruginosa invasion.a Transfection of human corneal epithelial cells with an antagomir of miR-762 (antago-762) decreased the expression of miR-762 relative to a control (scrambled) antagomir, and b increased the expression of Rab5a mRNA, a predicted target of miR-762. c Antago-762 also increased the expression of RNase7 and ST-2 mRNA suggesting that miR-762 negatively regulates these innate defense genes. Antago-762 did not affect genes encoding hBD-2 and hBD-3. d The antagomir did not affect epithelial susceptibility to P. aeruginosa invasion. Gene expression was measured by real-time PCR, and P. aeruginosa invasion by using gentamicin exclusion assays. In each experiment, cells were used at 48 h after transfection. For invasion assays, epithelia were challenged at 72 h with 104 cfu invasive P. aeruginosa strain 6294 for 3 h followed by 1 h gentamicin treatment (see Methods). (* Significant difference vs. respective controls, t-Test).

Mentions: We have previously shown that tear fluid upregulates epithelial genes encoding RNase7 and ST-2, and that these innate defense factors can protect epithelial cells against bacterial internalization [9]. Since miR-762 was the most profoundly upregulated microRNA by bacterial antigens in tear fluid treated cells (Fig. 1), we tested if miR-762 could influence epithelial expression of genes encoding RNase7 and/or ST-2, and/or affect bacterial internalization. Corneal epithelial cells were transfected with an antagomir to miR-762 (Antago-762) for 48 h under baseline conditions, i.e. without tear fluid or bacterial antigen exposure, to reduce miR-762 expression, and in turn, affect mRNA levels of genes targeted by this microRNA. As expected, Antago-762 effectively reduced epithelial expression of miR-762 at 48 h by ∼50% relative to a scrambled control (Fig. 2 a, p < 0.05, t-Test). Accordingly, Antago-762 increased expression of Rab5a mRNA, a predicted target of miR-762 (based upon analysis of predicted targets for miR-762 using microRNA.org), by ∼75% relative to the scrambled control (Fig 2 b, p < 0.05, t-Test). Interestingly, Antago-762 also increased gene expression of RNase7 (by ∼80%) and ST-2 (by ∼58%) (Fig. 2 c, p < 0.05, t-Test for each), suggesting that miR-762 negatively regulates the expression of genes encoding these innate defense factors. Antago-762 did not affect the expression of genes encoding hBD-2 and hBD-3 (Fig. 2 c), two other antimicrobial peptides expressed by these epithelia [32], [33], nor did it affect bacterial internalization by these epithelial cells (Fig. 2 d).


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)

The effect of a miR-762 antagomir on gene expression in human corneal epithelial cells and on P. aeruginosa invasion.a Transfection of human corneal epithelial cells with an antagomir of miR-762 (antago-762) decreased the expression of miR-762 relative to a control (scrambled) antagomir, and b increased the expression of Rab5a mRNA, a predicted target of miR-762. c Antago-762 also increased the expression of RNase7 and ST-2 mRNA suggesting that miR-762 negatively regulates these innate defense genes. Antago-762 did not affect genes encoding hBD-2 and hBD-3. d The antagomir did not affect epithelial susceptibility to P. aeruginosa invasion. Gene expression was measured by real-time PCR, and P. aeruginosa invasion by using gentamicin exclusion assays. In each experiment, cells were used at 48 h after transfection. For invasion assays, epithelia were challenged at 72 h with 104 cfu invasive P. aeruginosa strain 6294 for 3 h followed by 1 h gentamicin treatment (see Methods). (* Significant difference vs. respective controls, t-Test).
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pone-0057850-g002: The effect of a miR-762 antagomir on gene expression in human corneal epithelial cells and on P. aeruginosa invasion.a Transfection of human corneal epithelial cells with an antagomir of miR-762 (antago-762) decreased the expression of miR-762 relative to a control (scrambled) antagomir, and b increased the expression of Rab5a mRNA, a predicted target of miR-762. c Antago-762 also increased the expression of RNase7 and ST-2 mRNA suggesting that miR-762 negatively regulates these innate defense genes. Antago-762 did not affect genes encoding hBD-2 and hBD-3. d The antagomir did not affect epithelial susceptibility to P. aeruginosa invasion. Gene expression was measured by real-time PCR, and P. aeruginosa invasion by using gentamicin exclusion assays. In each experiment, cells were used at 48 h after transfection. For invasion assays, epithelia were challenged at 72 h with 104 cfu invasive P. aeruginosa strain 6294 for 3 h followed by 1 h gentamicin treatment (see Methods). (* Significant difference vs. respective controls, t-Test).
Mentions: We have previously shown that tear fluid upregulates epithelial genes encoding RNase7 and ST-2, and that these innate defense factors can protect epithelial cells against bacterial internalization [9]. Since miR-762 was the most profoundly upregulated microRNA by bacterial antigens in tear fluid treated cells (Fig. 1), we tested if miR-762 could influence epithelial expression of genes encoding RNase7 and/or ST-2, and/or affect bacterial internalization. Corneal epithelial cells were transfected with an antagomir to miR-762 (Antago-762) for 48 h under baseline conditions, i.e. without tear fluid or bacterial antigen exposure, to reduce miR-762 expression, and in turn, affect mRNA levels of genes targeted by this microRNA. As expected, Antago-762 effectively reduced epithelial expression of miR-762 at 48 h by ∼50% relative to a scrambled control (Fig. 2 a, p < 0.05, t-Test). Accordingly, Antago-762 increased expression of Rab5a mRNA, a predicted target of miR-762 (based upon analysis of predicted targets for miR-762 using microRNA.org), by ∼75% relative to the scrambled control (Fig 2 b, p < 0.05, t-Test). Interestingly, Antago-762 also increased gene expression of RNase7 (by ∼80%) and ST-2 (by ∼58%) (Fig. 2 c, p < 0.05, t-Test for each), suggesting that miR-762 negatively regulates the expression of genes encoding these innate defense factors. Antago-762 did not affect the expression of genes encoding hBD-2 and hBD-3 (Fig. 2 c), two other antimicrobial peptides expressed by these epithelia [32], [33], nor did it affect bacterial internalization by these epithelial cells (Fig. 2 d).

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