MiR-146a down-regulates inflammatory response by targeting TLR3 and TRAF6 in Coxsackievirus B infection

Figures

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  FIGURE 1.. MiR-146a abundance in the CVB3-infected cells. (A,B) Primary cardiac fibroblasts isolated from Balb/c mouse were infected with CVB3 (MOI = 5). Total RNA was collected at 24 and 48 h p.i. and subjected to miRNA array detection. (C–E) HeLa cells were infected with CVB3 (MOI = 1). The abundances of miR-146a, miR-21, and miR-186 at 0, 6, 12, 24, and 36 h p.i. were detected by RT-qPCR with the ΔΔCT method. The abundance of miRNAs was normalized by U6 (miRNAs). Error bars represent standard deviation (SD), n = 4.

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  FIGURE 2.. 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.

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  FIGURE 3.. Validation of miR-146a targeting to TLR3 and TRAF6. HeLa cells were transfected with miR-146a mimics, AMO-146a, and scramble RNAs, respectively. A group of the transfected cells were treated with LPS at 24 h posttransfection. Total protein was extracted at 24 h posttransfection or 6 h after the LPS treatment. The protein levels of TLR3 (A,C) and TRAF6 (B,D) were detected by western blotting. GAPDH was used as a loading control. Error bars represent the SD, n = 3.

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  FIGURE 4.. Effect of miR-146a on NF-κB translocation. HeLa cells were first treated with miR-146a mimics, AMO-146a, and scramble RNAs, respectively. The cells were treated with LPS at 18 h and harvested 24 h after the transfection. (A–D) Total, cytoplasmic, and nuclear portions of the p65 subunit of NF-κB were analyzed by western blotting. Total and cytoplasmic proteins were normalized using β-actin, whereas histidine H3 was used to normalize nuclear protein. (E,F) Detection of NF-κB activation with luciferase reporter assay. pNF-κB-Luc (Stratagene) is a plasmid in which Firefly luciferase (Luc) expression is modulated by an NF-κB response element. NF-κB activation can be measured by the level of Luc expression. HeLa cells were transfected with pNF-κB-Luc, pRL-TK, and various RNAs. The expression of Luc and RLuc in these cells was detected with dual luciferase reagents at 24 h posttransfection. (G,H) The mRNA and protein of IL-6 and TNF-α were detected with RT-qPCR and western blotting, respectively. Error bars represent SD, n = 3.

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  FIGURE 5.. The expression of TLR3 and TRAF6 in the CVB3- and EV71-infected cells. HeLa cells were treated with AMO-146a or scramble RNA. CVB3 and EV71 were infected at 12 h posttransfection, respectively. TLR3 and TRAF6 were detected with western blotting in the cells with CVB3 infection (A–C) and EV71 infection (D–F). TRAF6 and TLR3 were normalized by GAPDH. Error bars represent SD, n = 3.

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  FIGURE 6.. The TLR3-NF-κB pathway is inhibited by miR-146a in CVB3 infection. HeLa cells were treated with AMO-146a and scramble RNA, respectively. These cells were infected with CVB3 12 h posttransfection. (A) Total, cytoplasmic, and nuclear proteins were extracted 24 h p.i. The p65 subunit of NF-κB was detected with western blotting. The cytoplasmic fraction was normalized by β-actin, and the nuclear fraction was normalized by histidine H3. (B,C) The mRNA abundance of IL-6 and TNF-α were quantified by RT-qPCR. Error bars represent SD, n = 3.

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  FIGURE 7.. The putative role of miR-146a in CVB infection. CVB3 triggers an inflammatory response by activating NF-κB through multiple inductions (dashed lines). The activated NF-κB translocates to the nucleus and binds to the genomic locus of miR-146a in chromosome 5 to initiate the transcription of the downstream RNA including primary miR-146a. After a mature process, miR-146a conversely suppresses the NF-κB pathway by doubly blocking TLR3 and TRAF6. Through the negative feedback by miR-146a, the inflammation caused by CVB can be precisely controlled to an adequate level so that the viruses can be eliminated but the tissue is not overinjured.