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 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

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

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

Primers of mouse IL1a 3′-UTR and its mutated fragments.

<p>Primers of mouse IL1a 3′-UTR and its mutated fragments.</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

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