17β-Estradiol Attenuates LPS-Induced Macrophage Inflammation In Vitro and Sepsis-Induced Vascular Inflammation In Vivo by Upregulating miR-29a-5p Expression

Figure 4

miR-29a-5p targets NLRP3. (a) Prediction of the miR-29a-5p binding site at NLRP3 3-UTR. The red color indicates the sequence of the mutant miR-29a-5p binding site. (b) RAW 264.7 cells were transfected with the different concentrations of the miR-29a-5p mimic or mimic-ctl, and the miR-29a-5p level was detected by qRT-PCR. Relative expression of miR-29a-5p was presented after normalizing to U6 (; ). vs. the mimic-ctl group. (c) RAW 264.7 cells were transfected with the different concentrations of the miR-29a-5p inhibitor or inhibitor-ctl. qRT-PCR evaluated the expression of miR-29a-5p. Relative expression of miR-29a-5p was presented after normalizing to U6 (; ). vs. the inhibitor-ctl group. (d) Luciferase reporter assays were performed in 293A cells cotransfected cells with the miR-29a-5p mimic and wt or mutant (mut) NLRP3 3-UTR-luciferase reporter. vs. the mimic-ctl group. (e) Luciferase reporter assays in 293A cells transfected with the constructs containing the wt or mutant NLRP3 3-UTR after treatment with the inhibitor-ctl or miR-29a-5p inhibitor. vs. the inhibitor-ctl group. (f) RAW 264.7 cells were transfected with indicated constructs for 24 h; NLRP3 expression was detected by Western blotting. Left panel: representative blot from three independent experiments. Right panel: quantitation of NLRP3 normalized to β-actin. vs. the mimic-ctl group. ^### vs. the inhibitor-ctl group.

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