Pharmacological studies of the mechanism and function of interleukin-1beta-induced miRNA-146a expression in primary human airway smooth muscle.

BACKGROUND: Despite the widespread induction of miR-146a during the innate immune response little is known regarding its biogenesis, function and mechanism. We have therefore examined the role of miR-146a during the interleukin (IL)-1beta-stimulated IL-6 and IL-8 release and proliferation in primary human airway smooth muscle (HASM) cells. METHODS: HASM cells were isolated from human lung re-section, cultured to a maximum of 3 - 6 passages and then exposed to IL-1beta. miR-146a expression were determined by qRT-PCR, IL-6 and IL-8 release by ELISA and proliferation using bromodeoxyuridine incorporation. The role of NF-kappaB and the MAP kinase pathways was assessed using pharmacological inhibitors of IKK2 (TPCA-1), JNK (SP600125), p38 MAP kinase (SB203580) and MEK-1/2 (PD98059). miR-146a function was determined following transfection of HASM with inhibitors and mimics using Amaxa electroporation. RESULTS: IL-1beta induced a time-dependent and prolonged 100-fold induction in miR-146a expression, which correlated with release of IL-6 and IL-8. Exposure to IL-1beta had no effect upon HASM proliferation. Pharmacological studies showed that expression of primary miR-146a was regulated at the transcriptional levels by NF-kappaB whilst post-transcriptional processing to mature miR-146a was regulated by MEK-1/2 and JNK-1/2. Functional studies indicated that IL-1beta-induced miR-146a expression does not negatively regulate IL-6 and IL-8 release or basal proliferation. However, inhibition of IL-1beta-induced IL-6 and IL-8 release was observed at the super-maximal intracellular miR-146a levels obtained by transfection with miR-146a mimics and indicates that studies using miRNA mimics can produce false positive results. Mechanistic studies showed that in the presence of super-maximal levels, the action of miR-146a mimics was mediated at a step following IL-6 and IL-8 mRNA transcription and not through down-regulation of IL-1 receptor associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6) protein expression, two predicted miR-146a targets involved in IL-1beta signalling. CONCLUSIONS: We have shown that IL-1beta-induced miR-146a expression in HASM and that this was regulated at the transcriptional level by NF-kappaB and at the post-transcriptional level by the MEK-1/2 and JNK-1/2. Unlike previous reports, studies using miRNA inhibitors showed that miR-146a expression did not regulate IL-6 and IL-8 release or proliferation and suggest miR-146a function and mechanism is cell-type dependent.