Former scientific studies in HASM cells have shown that exposure to IL 1B activates NF B and the MAP kinase pathways terminating at ERK 1/2, JNK 1/2 and p38 MAP kinase. Consequently, established pharmacological inhibitors that had previ ously been proven to attenuate IKK2 and MAP kinase exercise in HASM had been utilized to examine the role of these intracellular pathways. Significantly, these studies indicated that miR 146a was regulated at the two the transcriptional and submit transcriptional level. As previ ously reported, we showed that preliminary transcription of key miR 146a was mediated as a result of activation of NF B. Additionally, we have demonstrated that ERK 1/2 and JNK 1/2 but not the p38 MAP kinase pathways regulate the processing of primary miR 146a to provide mature miR 146a. We attempted to confirm these pharmacological observations through the use of siRNA mediated knockdown of ERK 1/2 and JNK 1/2 but observed inhibition of IL 1B induced miR 146a produc tion while in the presence of handle siRNA.
Dicer is thought to cleave the precursor miRNA to provide the double stranded miRNA and in mixture with TRBP, is required for the loading of both siRNA and miRNAs in to the Ago2 containing RISC complex. We therefore specu late that transfected siRNA might possibly compete with precur order Roscovitine sor miR 146a for Dicer binding and by this route, siRNA could block the manufacturing of mature miR 146a. Signifi cantly, competition amongst siRNA and miRNA has not long ago been demonstrated by Khan A et al.. More than all, this is actually the very first report demonstrating a purpose for ERK 1/ 2 and JNK 1/2 pathways inside the regulation of miR 146a biogenesis and although the mechanism is presently unknown, we speculate that these MAP kinases might regulate proteins involved with miRNA processing or stabil ity.
Examination in the effect LY500307 of those MAP kinase inhibi tors upon generation of inflammatory mediators showed that IL six release was mediated through NF B, ERK 1/2 and p38 MAP kinase while IL eight release was mediated via NF B and ERK 1/2. Considerably, due to the fact neither IL six nor IL eight release is influenced by the JNK 1/2 inhibitor, it had been pos sible to utilize the JNK 1/2 inhibitor to examine the perform of miR 146a for the duration of IL 1B induced IL six and IL eight release. Previous investigations in alveolar epithelial cells, monocytes and macrophages have proven that improved ranges of miR 146a negatively regulate the release of inflammatory mediators. Transfection with miR 146a mimics, which triggered a 3000 fold boost in cellular miR 146a ranges, could also inhibit IL 1B induced IL 6 and IL 8 release in HASM cells. Nevertheless, we showed the 100 fold increase in miR 146a expres sion following IL 1B stimulation is insufficient to inhibit IL 6 and IL 8, because attenuation of miR 146a exercise or blocking miR 146a expression had no signifi cant effect on cytokine release.