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. 2018 Aug 22;9:1916. doi: 10.3389/fimmu.2018.01916

Figure 5.

Figure 5

MiR-223 inhibits IL-1β release by targeting NLRP3. (A) Immunohistochemical staining of NLRP3, scale bar, 50 μm. (B) Conservation of the miR-223 target sequence in NLRP3 among different species (Upper), and conservation of the sequence of miR-223 among different species (Lower). (C) Schematic diagram showing dual-luciferase reporter constructs harboring the 3′UTR of NLRP3 with the putative miR-223 binding site. The lower shows the alignment of miR-223 and its target site in the 3′UTR of NLRP3. Six mutated nucleotides of the target site are underlined. 293T cells were cotransfected with agomiR-223, antagomiR-223, or the corresponding control oligonucleotide (mimic-NC, inhibitor-NC) together with a wild-type or mutated NLRP3 3'UTR dual-luciferase reporter plasmid; luciferase activity was normalized by the ratio of firefly and Renilla luciferase signals. (D) BEND cells were transfected with agomiR-223, antagomiR-223, or the corresponding control oligonucleotide (miR-NC, anti-NC), and then, the NLRP3 protein levels were determined after 24 h by Western blotting. BEND cells transfected with NLRP3 siRNA or the corresponding control oligonucleotide (si-NC) for 24 h and induced by LPS (1 μg/mL) for 2 h. (E) The NLRP3 level was determined by qPCR. (F) The concentration of IL-1β in BEND cell supernatant was detected by ELISA. (G) The mRNA of chemokines CXCL1, CXCL2 was determined by qPCR. Data represent three independent experiments and are presented as the mean ± S.E.M (error bars). Two tailed, student t-test, *P < 0.05; **P < 0.01.