Potential effects and mechanisms of the regulation of inflammation by miR-181a.

<p>Potential effects and mechanisms of the regulation of inflammation by miR-181a.</p

(A and B) Effects of transient transfection with miR-181a expression vector on the levels of ILa, IL1b, IL6, and TNFa in PMA/LPS-induced THP-1 cell lysis solution and culture media.

<p>Data are expressed as mean ± SD (n = 3), *<i>P</i><0.05, **<i>P</i><0.01 vs. CMV controls. ‘SC’, pCMV-pre-miR-181a recombinant THP-1 cells; ‘CMV’, pCMV recombinant THP-1 cells (negative controls). (C and D) Effects of si-IL1a on the mRNA levels of IL1a and IL1b in LPS-induced Raw264.7 and PMA/LPS-induced THP-1 cells. (E and F) Effects of si-IL1a on the levels of IL1a, IL1b, IL6, and TNFa in LPS-induced Raw264.7 and PMA/LPS-induced THP-1 cells. Data are expressed as mean ± SD (n = 3), *<i>P</i><0.05, **<i>P</i><0.01 vs. negative control (NC). ‘siNC’ is a single-stranded nucleic acid used as negative control for siIL1a.</p

Sensitive Conjugated-Polymer-Based Fluorescent ATP Probes and Their Application in Cell Imaging

Three cationic conjugated polyelectrolytes (CPEs) with a common poly­(<i>p</i>-phenylene ethynylene terthiophene) backbone and side chains of different lengths, named as PPET3-N1, PPET3-N2, and PPET3-N3, were designed and synthesized. The UV–vis absorption and fluorescence spectra of the polymers vary strongly with solvent composition, suggesting that the polymers are strongly aggregated in H₂O. In addition, the spectroscopic properties of the polymers are affected by small-molecule ATP, characterized by significant fluorescence intensity decreases and red shifts of their absorption bands. Further application of these polymers in cell imaging was studied by confocal fluorescence microscopy, which demonstrated that all of the polymers were localized on the cell membrane and partially inside of cells and that the staining effect gradually increased with the length of the polymer side chains. On the basis of the low cytotoxicity and efficient quenching of PPET3-N2 by ATP, the dose and time effects of ATP on PPET3-N2 imaging were studied, and the results indicated that this polymer might have potential in cell imaging for ATP semiquantification in vivo

Effects of miR-181a mimics and inhibitors on the levels of IL1a, IL1b, IL6, and TNFa in IL1a-induced THP-1 cell lysis solution (A) and culture medium (B).

<p>Data are expressed as mean ± SD (n = 3), *<i>P</i><0.05, **<i>P</i><0.01 vs. negative control (NC), ^#<i>P</i><0.05 vs. miRNA inhibitor negative control (NC-i). ‘NC-i’ is a single-stranded nucleic acid used as a negative control for miR-181a inhibitors (181a–i). IL1a + and IL1a- indicate that the cells were treated with (at a final concentration of 0.1 ng/ml) and without exogenous IL1a, respectively.</p

Effects of miR-181a mimics and inhibitors on the productions of reactive oxygen species (ROS) in PMA/LPS-induced TPH-1 cells.

<p>Data are expressed as mean ± SD (n = 3), **<i>P</i><0.01 vs. negative control (NC), ^##<i>P</i><0.01 vs. miRNA inhibitor negative control (NC-i). ‘NC-i’ is a single-stranded nucleic acid used as negative control for miR-181a inhibitors (181a–i). LPS+ and LPS− indicate cells treated with and without PMA/LPS, respectively.</p

miR-181a Regulates Inflammation Responses in Monocytes and Macrophages

<div><p>miR-181a has been presumed to target the 3′-untranslated regions (3′-UTR) of IL1a based on software predictions. miR-181a and IL1a have opposite expression levels in monocytes and macrophages in the inflammatory state. This led us to suspect that mir-181a has an important function in regulating inflammatory response by targeting IL1a. Fluorescence reporter assays showed that miR-181a effectively binds to the 3′-UTR of IL1a. The anti-inflammatory functions of miR-181a were investigated in lipopolysaccharides (LPS)-induced Raw264.7 and phorbol 12-myristate 13-acetate (PMA)/LPS-induced THP-1 cells. We found that miR-181a mimics significantly lowered IL1a expression levels in these cells and, interestingly, miR-181a inhibitors reversed this decrease. In addition, miR-181a mimics significantly inhibited increase in the levels of inflammatory factors (IL1b, IL6, and TNFa) in these cells. Furthermore, miR-181a mimics and inhibitors decreased and increased, respectively, production of reactive oxygen species in PMA/LPS-induced THP-1 cells. These results indicate that miR-181a regulates inflammatory responses by directly targeting the 3′-UTR of IL1a and down-regulating IL1a levels. Interestingly, we found that miR-181a inhibited production of inflammatory factors even in IL1a-induced THP-1 cells, suggesting that the anti-inflammatory effects of miR-181a possibly involves other targets in addition to IL1a. Thus, we provide the first evidence for anti-inflammatory effects of miR-181a mediated at least in part by down-regulating IL1a.</p> </div

Effects of miR-181a mimics and inhibitors on IL1a and IL1b mRNA levels in LPS-induced Raw264.7 (A) and PMA/LPS-induced THP-1 cells (B); effects of miR-181a mimics and inhibitors on ILa, IL1b, IL6, and TNFa in cell lysates and media of LPS-induced Raw264.7 (C and E) and PMA/LPS-induced THP-1 cells (D and F) assayed by ELISA.

<p>Data are expressed as mean ± SD (n = 3), **<i>P</i><0.01 vs. negative control (NC), ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. miRNA inhibitor negative control (NC-i). ‘NC-i’ is a single stranded nucleic acid used as negative control for miR-181a inhibitors (181a–i). ‘LPS + or −’, cells were treated with or without LPS in the case of Raw264.7 cells or PMA/LPS in the case of THP-1 cells.</p

Primers for energy metabolism genes.

<p>Primers for energy metabolism genes.</p

Effects of stable expression of miR-181a on production of inflammatory factors in LPS-induced Raw264.7 cells.

<p>(A) pCMV recombinant Raw264.7 cells (CMV, negative controls); (B) pCMV-pre-miR-181a recombinant Raw264.7 cells (SC); (C) endogenous miR-181a expression in SC (containing miR-181a expression vector) and CMV (containing negative control vector) cells; (D) Expression of IL1a in pCMV and pCMV-pre-miR-181a recombinant Raw264.7 cells assayed by Western blotting; (E and F) Levels of IL1a, IL1b, IL6, and TNFa in cell lysis solution and culture media of recombinant Raw264.7 cells transfected with pCMV and pCMV-pre-miR-181a. Data are expressed as mean ± SD (n = 3), *<i>P</i><0.05, **<i>P</i><0.01 vs. pCMV recombinant controls induced by LPS (CMV LPS+), ^##<i>P</i><0.01 vs. miRNA inhibitor negative control (NC-i). LPS+ and LPS− indicate that cells were treated with and without LPS, respectively.</p

Presumed binding sites for miR-181a in the 3′-UTR of mouse and human IL1a.

<p>The binding sites are highly conserved.</p