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. 2021 Nov 13;24(12):103441. doi: 10.1016/j.isci.2021.103441

Figure 4.

Figure 4

ss-miR-146a-5p downregulates IRAK-1 protein expression through TLR7 signaling and cellular proteasome activation

(A) Schematic diagram of in vivo (20 μg, i.p.) and in vitro (50 nM, BMDMs) treatment with ss miR-146a-5p and ds miR-146a duplex.

(B) Downregulation of Irak-1 transcript in the peritoneal cells after i.p. injection of or BMDMs treated with ss-miR-146a-5p, dd miR-146a duplex, mutants, or vehicle alone (Lipo) for 24 h; n = 4. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA.

(C) Pretreatment of BMDMs with ss-miR-146a-5p or ds miR-146a duplex desensitizes BMDMs to subsequent LPS, but not TNFα, stimulation. BMDMs were pretreated with ds miR-146a, ss miR-146a-5p, or the indicated mutants, all at 50 nM, for 16 h, washed, and then challenged with 10 ng/mL LPS or 10 ng/mL TNFα for 16 h n = 3. ∗p < 0.05, ∗∗p < 0.01, Welch t test.

(D and E) Time course of IRAK-1 protein (D) and transcript (E) downregulation after treatment with miR-146a mimics and TLR ligands. BMDMs were treated with Pam3cys (1 μg/mL), poly IC (0.5 μg/mL), LPS (0.1 μg/mL), R837 (0.5 μg/mL), ss-miR-146a-5p (50 nM), or ds miR-146a (50 nM) for 2, 4, or 24 h. At each time point, cellular IRAK-1 protein and mRNA (n = 3) were analyzed using western blot and qRT-PCR, respectively.

(F) Western blot analysis of IRAK-1 protein in WT and miR-146a−/− BMDMs treated with ss-miR-146a-5p, U→A mutant, or vehicles (Lipo) alone.

(G) Western blot analysis of IRAK-1 protein in miR-146a−/− BMDMs treated with Pam3cys (1 μg/mL), LPS (0.1 μg/mL), R837 (0.5 μg/mL), miR-145a-5p (50 nM), or miR-146a-5p (50 nM) for 24 h

(H) ds miR-146a duplex, but not ss miR-146a-5p, represses luciferase output of an IRAK-1-3′ UTR reporter assay. Irak-1-3′UTR luciferase reporter assay in the presence of scrambled ds miR-146a (ds146a scr), ds miR-146a (ds146a), or ss-miR-146a-5p (ss146a), all at 50 nM. The experiments were performed in triplicates and repeated twice. ∗∗p < 0.01, Student t test.

(I and J) Immunoprecipitation of Ago2 in miR-146a−/− BMDMs treated with ss miR-146a-5p and ds miR-146a duplex.

(K) miR-146a-5p associated with Ago2 protein. miR-146a−/− BMDMs were incubated with ss-miR146a-5p or ds miR-146a duplex (100 nM) for 24 h. The levels of miR-146a-5p associated with Ago2 were quantified using qRT-PCR following Ago2 IP (ss146a-IgG, n = 3; n = 4 for the rest groups). ∗p < 0.05, Student t test.

Data are represented as mean ± SD.