Research Paper Volume 14, Issue 8 pp 3633—3651

Figure 8. ET-1 suppresses production of NFkB, TNF-α and IL-6 by increasing miR-let-7g-5p expression. (A) Open-source software (TargetScan, miRDB, and miRWalk) sought to identify miRNAs that could possibly interfere with NFkB, IL-6 and TNF- α transcription. (B) Cells were incubated with melatonin (0-50 nM) for 24 h and miR-let-7g-5p expression was examined by qPCR. (C) Cells were pretreated with BQ123+BQ788, Ly294002, Akt inhibitor for 30 min, then stimulated with ET-1 for 24 h. miR-let-7g-5p expression was examined by qPCR. (D, E) Cells were transfected with the miR-let-7g-5p mimic and then treated with ET-1 (50 nM). TNF-α and IL-6 expression was evaluated by qPCR. The wild-type and mutant Ikbkb 3′-UTRs contained the miR-let-7g-5p binding site. (F) Cells were transfected with the miR-let-7g-5p mimic and then treated with ET-1 (50 nM). (G) Cells were transfected with 3′-UTR plasmids as indicated then stimulated with ET-1 dose concentration. Then, cells were transfected with indicated luciferase plasmids for 24 h then stimulated with ET-1 for 24 h. Relative luciferase activity was measured. Results are expressed as the mean ± SEM. ^*P < 0.05 compared with controls; ^#P < 0.05 compared with the melatonin-treated group.