Figure 5.

Extracellular miR-146a-5p downregulates IRAK-1 protein via TLR7→MyD88 signaling and proteasome activation

(A) miR-146a duplex-mediated IRAK-1 downregulation is preserved in TLR7^−/− BMDMs. Immunoblot analysis of IRAK-1 in WT and TLR7^−/− BMDMs treated with miR-146a duplex (ds146a, 50 nM) or scrambled control (146a scr, 50 nM) for 48 h. Experiment was performed in triplicates and repeated twice. ∗p < 0.05, ∗∗p < 0.01, versus scrambled miR-146a duplex (146 scr). Welch t test.

(B) ss-miR-146a-5p-mediated downregulation of IRAK-1 protein is abolished in TLR7^−/− BMDMs. Immunoblot analysis of IRAK-1 in WT and TLR7^−/− BMDMs treated with miR-146a-5p or the U→A mutant (50 nM). ∗∗p < 0.01 versus U→A mutant in WT BMDMs. Welch t test. Experiments were done in duplicates or triplicates and repeated twice.

(C) Immunoblot analysis of IRAK-1 in WT and TLR7^−/− BMDMs treated with ss miR-145a-5p (50 nM) or vehicle (Lipo). ∗∗p < 0.01 versus WT Lipo. Welch t test. Experiments were done in triplicates and repeated twice.

(D) IRAK-1 downregulation by ss-miR-146-5p requires MyD88 but not Trif. Immunoblot analysis of IRAK-1 in WT, MyD88^−/−, and Trif^−/− BMDMs treated with miR-146a-5p (50 nM). ∗p < 0.05 versus WT Lipo, Welch t test. Experiments were repeated twice.

(E) Proteosome-mediated IκB degradation after LPS stimulation is inhibited by MG132. IκB-α immunoblot in BMDMs stimulated with LPS (100 ng/mL) in the absence or presence of proteasome inhibitor MG-132 (1 μM). ∗p < 0.05, Welch t test.

(F) Rapid miR-146a-5p-mediated IRAK-1 downregulation is inhibited by MG132. IRAK-1 immunoblot in BMDMs stimulated with 50 nM miR-146a-5p in the absence or presence of proteasome inhibitor MG-132 (1 μM). ∗p < 0.05, ∗∗p < 0.01, Student t test.

Data are represented as mean ± SD.