Fig. 2
Analysis of signaling pathways and transcription factors involved in regulation of miR-155 and miR-146a expression. a and b U251 cells were infected with PCN033 or mock-infected in the presence of BAY11072, SP600125, U0126, and SB202190. The expression of miR-155 and miR-146a was detected by real-time qPCR. Data are presented as the mean + SD (n = 3/group). Statistical analysis was carried out by one-way ANOVA. c Schematic diagram of miR-155 genomic loci. A NF-κB binding site was predicted upstream of the MIR155HG transcription start site (+1). d Luciferase reporter assays were performed based on the co-transfection of a miR-155 promoter luciferase reporter (WT1), pRL-TK vector, and increasing concentrations of pcDNA3.1-p65 plasmid or empty vector control (pcDNA). Then, 24 h later, luciferase activity was measured, and the results were expressed as relative luciferase activity by normalizing firefly luciferase activity against Renilla luciferase activity, and results were recorded as the mean + SD from three replicate wells. e HEK-293T cells were co-transfected with a miR-155 promoter luciferase reporter or NF-κB mutation construct of the miR-155 promoter (NF-κB MUT1), along with pcDNA3.1-p65 plasmid. f Schematic diagram of miR-146a genomic loci. Two NF-κB binding sites were predicted upstream of the pri-miR-146a transcription start site (+1). g HEK-293T cells were co-transfected with a miR-146a promoter luciferase reporter (WT2), pRL-TK vector, and increasing concentrations of pcDNA3.1-p65 plasmid or empty vector control. h The miR-146a promoter luciferase reporter or NF-κB mutation construct of the miR-146a promoter (NF-κB MUT2), along with pcDNA3.1-p65 plasmid, were co-transfected into HEK-293T cells, and luciferase activity was measured. Error bars represented the SD calculated from the results of at least three independent experiments. Statistical analysis was carried out by one-way ANOVA. p < 0.05 was considered statistically significant; p < 0.01 and p < 0.001 indicated extremely significant differences