miR-146a directly downregulates IRAK1 and TRAF6 in macrophages after hypoxia/reoxygenation <i>in</i><i>vitro</i>.

<p>RAW264.7 cells were exposed to 1 or 3 h of hypoxia and followed by various periods (2, 4, or 6 h) of reoxygenation. (A) qPCR analysis of miR-146a expression. (B) Supernatants were collected to measure TNF-α by ELISA. (C) The levels of miR-146a were decreased in macrophages following 3 h of hypoxia and 6 h of reoxygenation. (D) qRT-PCR analysis and immunoblotting of IRAK1 and TRAF6 in macrophages after hypoxia/reoxygenation (H/R). RAW 264.7 cells were transfected with miR-146a mimic, miR-146a inhibitor or relative control. (E) The efficacy of transfection was determined by qRT-PCR. (F) The mRNA and protein expression of IRAK1 and TRAF6 were determined by qRT-PCR and Western blot, respectively. (G and H) The sequence alignment of miR-146a and its putative and mutated target sites of the mouse IRAK1 and TRAF6 mRNA 3′untranslated region (3′UTR) were shown. RAW 264.7 cells were transiently co-transfected with luciferase report vectors, and in the presence of miR-146a mimic. Luciferase activities were normalized to the activity of Renilla luciferase. Results are mean ± SD. <b>*</b>, P<0.05 and <b>**</b>,P<0.01,<b>***</b>,P<0.001 versus negative control. H/R, hypoxia/reoxygenation; M, miR-146a mimic; mNC, negative control miRNA of miR-146a mimic; I, miR-146a inhibitor; iNC, negative control miRNA of miR-146a inhibitor.