MiR-181a regulates inflammation responses in monocytes and macrophages

Abstract

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.

Figure 1. Presumed binding sites for miR-181a in the 3′-UTR of mouse and human IL1a.

The binding sites are highly conserved.

Figure 2. (A and B) miR-181a expression in LPS-induced Raw264.7 cells and PMA/LPS-induced THP-1 cells; (C and D) intracellular IL1a levels in LPS-induced Raw264.7 cells and PMA/LPS-induced THP-1 cells; (E) effects of miR-181a mimics on chemiluminescence values in Cos-7 cells (transfected with pRLTK containing L1a-3′-UTR or its mutated variant).

Data are expressed as mean ± SD (n = 3), **P<0.01 vs. values at 0 h (A–D), **P<0.01 vs. negative control (NC, E).

Figure 3. 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.

Data are expressed as mean ± SD (n = 3), **P<0.01 vs. negative control (NC), ^# P<0.05, ^## P<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.

Figure 4. Effects of miR-181a mimics and inhibitors on ILa levels in cell lysis solution of LPS-induced Raw264.7 (A) and PMA/LPS-induced THP-1 cells (B) assayed by Western blot.

Data are expressed as mean ± SD (n = 3–5), **P<0.01 vs. negative control (NC), ^## P<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). Effects of miR-181a precursors (pre-miR-181a) and its inhibitors on ILa, IL1b, IL6, and TNFa in cell lysates and culture media of PMA/LPS-induced THP-1 cells (C and D) assayed by ELISA. Data are expressed as mean ± SD (n = 3), *P<0.05, **P<0.01 vs. negative control (pre-NC), ^# P<0.05 vs. miRNA inhibitor negative control (pre-NC-i). ‘pre-NC-i’ is a single-stranded nucleic acid used as negative control for miR-181a inhibitors (pre-miR-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.

Figure 5. Effects of stable expression of miR-181a on production of inflammatory factors in LPS-induced Raw264.7 cells.

(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), *P<0.05, **P<0.01 vs. pCMV recombinant controls induced by LPS (CMV LPS+), ^## P<0.01 vs. miRNA inhibitor negative control (NC-i). LPS+ and LPS− indicate that cells were treated with and without LPS, respectively.

Figure 6. (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.

Data are expressed as mean ± SD (n = 3), *P<0.05, **P<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), *P<0.05, **P<0.01 vs. negative control (NC). ‘siNC’ is a single-stranded nucleic acid used as negative control for siIL1a.

Figure 7. 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).

Data are expressed as mean ± SD (n = 3), *P<0.05, **P<0.01 vs. negative control (NC), ^# P<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.

Figure 8. Effects of miR-181a mimics and inhibitors on the productions of reactive oxygen species (ROS) in PMA/LPS-induced TPH-1 cells.

Data are expressed as mean ± SD (n = 3), **P<0.01 vs. negative control (NC), ^## P<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.

Figure 9. Potential effects and mechanisms of the regulation of inflammation by miR-181a.