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. 2019 Dec 5;135(3):167–180. doi: 10.1182/blood.2019002045

Figure 2.

Figure 2.

C-miR146a mimic targets upstream regulators of NF-κB signaling. (A) C-miR146a inhibits IRAK1 and TRAF6 expression through a sequence-dependent, on-target effect. HEK293T cells were incubated for 24 hours with 500 nM of C-miR146a, control C-scrRNA, or miR146a alone, and then transfected with WT or mutated IRAK1 or TRAF6 3′UTR luciferase reporter and control Renilla luciferase plasmids. The reporter luciferase activities were evaluated after 48 hours. (B) Reduced protein levels of IRAK1 and TRAF6 in C-miR146a–treated mouse macrophages and human leukemia cells. IRAK1 and TRAF6 protein levels were assessed by using western blot in RAW264.7 and MDSL cells after 48 hours of incubation with 500 nM of the indicated oligonucleotides. Shown are representative results; band intensities were quantified with normalization to β-actin as a loading control. (C) miR-146a mimic delivery prevents nuclear translocation of NF-κB. RAW264.7 cells stably expressing p65-eGFP fusion protein were incubated overnight with 500 nM of C-miR146a or control C-scrRNA and then stimulated with 100 ng/mL of LPS for 4 hours. Translocation of NF-κB/p65 (green) into nuclei (blue) was visualized by using confocal microscopy. (D) C-miR146a inhibits NF-κB DNA binding in target myeloid cells, RAW264.7, or MDSL. Cells were incubated with 500 nM of C-miR146a, C-anti-miR146a, or control C-scrRNA for 48 hours. The NF-κB DNA binding was assessed in nuclear extracts and verified by using p65-specific antibody supershift. Representative blots (left/middle) and the quantification of band intensities combined from 3 experiments (right). (E-F) C-miR146a reduces transcriptional activity of NF-κB in macrophages. RAW-Blue (E) or RAW264.7 (F) cells were incubated overnight with 500 nM of C-miR146a or control C-scrRNA and then stimulated with 100 ng/mL of LPS for 4 hours. The expression of the NF-κB–dependent reporter gene (panel E) or IL-6 secretion (panel F) was assessed by using Quanti-Blue assay or ELISA, respectively. Shown are representative results obtained in 3 independent experiments; means ± SEM. ***P < .001 and *P < .05 compared to untreated groups or as indicated. RLU, relative luciferase unit; SEAP, secreted alkaline phosphatase.