TRAF6 is a direct target of miR-146b-5p and knock-down of miR-146b-5p increased TRAF6 expression in human tTregs (n = 3). To assess whether human miR-146b-5p targets TRAF6, HEK293 cells were transduced with plasmids carrying wild-type (WT) or mutant (MUT) 3′ UTR sequences from TRAF6 linked to a luciferase reporter gene. Cells were also transfected with a Renilla luciferase reporter construct for normalization. (A) Three software packages (targetscan.org, MIRDB, and microRNA.org) were used to predict the potential target mRNAs of miR-146b-5p; TRAF6 was involved in tTreg function with highest probability. (B) Schematic representation of the miR-146b-5p target sequence within the 3′ UTR of TRAF6. Two nucleotides (complementary to nucleotides 6 and 8 of miR-146b-5p) were mutated in the 3′ UTR of TRAF6. The numbers indicate the positions of the nucleotides in the reference WT sequences. (C) Activity of the luciferase gene linked to the WT or MUT 3′ UTR of TRAF6. Luciferase activity was measured after 48 hr. The mean of the results from the cells transfected by control vector was set as 100%. The data are mean and standard deviation (SD) of separate transfections (n = 3). Naive peripheral blood tTregs were sort-purified, expanded in vitro, and treated with or without miR-146b antagomir or scrambled RNA as previously described. After treatment, cultured cells were assessed for TRAF6 mRNA and protein expression by RT-PCR or flow cytometry (D and E, respectively). Values indicate mean ± SEM of these experiments. *P < .05; **P < .01.