Figure 2: Regulation of miR-146a and the effect of EV71-induced miR-146a on interferon production.

(a) Schematic organization of miR-146a. AP1 (c-jun/c-fos) BS were predicted by intersection of TRANSFAC, PROMO and JASPAR software. (b) AP1 was upregulated by EV71 infection (n=3). MI, mock infection. *P-value <0.05 compared with mock infection. (c,d) c-jun and c-fos enhance the promoter activity and expression of miR-146a. RD cells were transfected with the c-jun or c-fos overexpressing vector and assayed for promoter activity (n=4) (c) and miR-146a expression (n=3) (d). MT, mock transfection. *P-value <0.05 as compared with vector control. (e) Determination of AP1 BS within miR-146a promoter. AP1 core sequence mutants are indicated in a. The transcriptional activities of miR-146a mutant promoters were determined in the presence of AP1 (n=4). *P<0.05 as compared with the wild-type control. (f) EV71 activated miR-146a promoter harbouring a natural context but not a mutant one (n=4). (g) Silencing of AP1 decreased virus-induced miR-146a expression and restored IRAK1 and TRAF6. (h) Suppression of IRAK1 and TRAF6 was restored by antagomiR-146a. (i) Inhibition of IFNβ promoter activity was attenuated by antagomiR-146a. IFNβ promoter was constructed and assayed by promoter assays (n=4). *P-value <0.05 as compared with antagomiR-N.C. (j) AntagomiR-146a eliminated the suppression of IFNβ expression in EV71 infection. IFNβ expressions were determined by real-time RT–PCR (n=3). MI, mock infection. *P-value <0.05 as compared with antagomiR-N.C. All data presented are mean±s.d. and all P-values are calculated by Student’s t-test.