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. 2023 Feb 27;14:1095457. doi: 10.3389/fimmu.2023.1095457

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Copyright © 2023 Yang, Zhao, Ji, Wang, Liu, Bao, Liu, Jiang, Zhang and Tang

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Circ_0075723 functions as a sponge for miR-155-5p in THP-1. (A) Schematic showing the predicted miR-155-5p sites in circ_0075723. (B) qRT-PCR analysis of the expression of miR-155-5p in CD14+ monocytes among the pneumonia-induced sepsis, pneumonia without sepsis and healthy control group. Each group has 4 samples. Data are presented as means ± SD; significant difference was identified with one-way ANOVA; ***P < 0.001 vs. Control or Pneumonia. (C) qRT-PCR analysis of the expression of miR-155-5p in THP-1 cells primed with different doses of LPS (0.1, 0.5 and 1 μg/ml) for 4 h and stimulated with nigericin (10 μM) for 2h. Data are presented as means ± SD; significant difference was identified with Student t-tests. **p < 0.01 vs. PBS; ***p < 0.001 vs. PBS. (D) THP-1 cells were transfected with vector or shRNA scrambled control (shRNA NC) or were transfected with circ_0075723-overexpressing lentivirus plasmids (OE-circ_0075723), sh-circ_0075723-expressing lentivirus plasmids (sh-circ_0075723), vector or shRNA scrambled control (shRNA NC) and then were primed with LPS (1 μg/ml) for 4 h and stimulated with nigericin (10 μM) for 2h. qRT-PCR analysis of the expression of miR-155-5p in THP-1 cells. Data are presented as means ± SD; significant difference was identified with Student t-tests. **p < 0.01 vs. vector; ***p < 0.001 vs. shRNA NC; #p < 0.05 vs. LPS/nigericin + Vector; &p < 0.05 vs. LPS/nigericin + shRNA NC. (E) RNA pull-down analysis of the interaction between miR-155-5p and circ_0075723. Data are presented as means ± SD; significant difference was identified with Student t-tests. ***p < 0.001 vs. NC group. (F) Dual-luciferase reporter assay was performed to validate the association between miR-155-5p and circ_0075723. Data are presented as means ± SD; significant difference was identified with one-way ANOVA. **p < 0.01 vs. mimic NC group. THP-1 cells were transfected with vector + mimic scrambled control (mimic NC) or vector + miR-155-5p mimic or were transfected with vector + mimic NC, vector + miR-155-5p mimic, mimic NC + circ_0075723 lentivirus plasmids (circ_0075723 OE), or miR-155-5p mimic + circ_0075723 OE and then were primed with LPS (1 μg/ml) for 4 h and stimulated with nigericin (10 μM) for 2h. (G) Western blot analysis of TLR4, NLRP3, ASC1, caspase1, cleaved caspase-1, Pro-IL1β, IL-1β, GSDMD and GAPDH in THP-1 cells. (H) ELISA of IL-18 and IL-1β in THP-1 supernatant. Data are presented as means ± SD; significant difference was identified with Student t-tests. *p < 0.05 vs. vector + mimic NC; **p < 0.01 vs. vector + mimic NC; ***p < 0.001 vs. vector + mimic NC; ##p < 0.01 vs. LPS/nigericin + vector + mimic NC; ###p < 0.001 vs. LPS/nigericin + vector + mimic NC; &&p < 0.01 vs. LPS/nigericin + circ_0075723-OE + mimic NC.