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.
Airway Disease, National Heart and Lung Institute, Imperial College London, London, UK.
Hanna M
HM
Larner-Svensson
Andrew E
AE
Williams
Eleni
E
Tsitsiou
Mark M
MM
Perry
Xiaoying
X
Jiang
Kian F
KF
Chung
Mark A
MA
Lindsay
D049109:Cell Proliferation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D007113:Immunity, Innate; D053583:Interleukin-1beta / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008168:Lung / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D016133:Polymerase Chain Reaction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C508606:CXCL8 protein, human; C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; C482128:MAP2K2 protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human