TLR3 and TRAF6 are the targets of miR-146a. (A,B) There are two sites in TLR3 mRNA and three sites in TRAF6 mRNA that perfectly match to the seed sequence of miR-146a. (C–E) HeLa cells were transfected with miR-146a mimics and infected with CVB3 (MOI = 1) 24 h posttransfection. The mRNA abundances of TLR3 and TRAF6 were quantified by RT-qPCR with the ΔΔCT method. (F–H) HeLa cells were transfected with AMO-146a and infected with CVB3 (MOI = 1) 24 h posttransfection. The mRNA abundances of TLR3 and TRAF6 were quantified by RT-qPCR with the ΔΔCT method. The mRNA abundances of TLR3 and TRAF6 were normalized by GAPDH mRNA, whereas miR-146a was normalized by U6 snRNA. Error bars represent SD, n = 4. (I–K) Two putative targets of TLR3 (1985–1992 nt and 2885–2891 nt) were inserted separately into the 3′UTR sequence of Renilla luciferase (RLuc) gene in plasmid pGL4.75. The modified pGL4.75 was transfected with Firefly luciferase (Luc)–expressing plasmid pGL4.17 and 40 or 60 pmol of miR-146a simultaneously to HeLa cells. Luciferase expression in these cells was detected at 24 h posttransfection. The level of RLuc in each well was normalized by Luc. Error bars represent SD, n = 3.
