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MEG3-4 is a miRNA decoy that regulates IL-1β abundance to initiate and then limit inflammation to prevent sepsis during lung infection

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Science Signaling  26 Jun 2018:
Vol. 11, Issue 536, eaao2387
DOI: 10.1126/scisignal.aao2387
  • Fig. 1 MEG3-4 is in a decreased abundance in mouse lungs after bacterial infection.

    (A) C57BL/6J mice were intranasally infected with 5 × 106 colony-forming units (CFU) of PAO1 for 24 hours. Lungs were lysed to assess the profiling of lncRNAs using the inflammatory response–based RT2 lncRNA primer array. Yellow dots indicate increased lncRNAs, and blue dots indicate decreased lncRNAs (more than twofold change and P ≤ 0.05 by Student’s t test). Data are from n = 2 biological replicates. (B) Schematic of MEG3 transcripts (http://vega.archive.ensembl.org/Mus_musculus/Transcript). Yellow, general MEG3 isoform probes; blue, specific MEG3-4 primers and probes. bp, base pair. (C) Northern blots of MEG3 transcripts in the lungs from control (CTRL) or PAO1-infected mice. 18S and 28S ribosomal RNAs (rRNAs) were used as loading controls. (D) In situ analysis of MEG3-4 abundance in the lungs from mice described in (C) with a digoxigenin (DIG)–labeled MEG3-4 probe (brown stain). Nuclei were counterstained by hematoxylin (blue). Scale bar, 100 μm. (E) qRT-PCR analysis of MEG3-4 expression in the lung, liver, spleen, and kidney from PAO1-infected mice. (F and G) qRT-PCR (F) and Northern blots (G) of MEG3-4 transcripts in the lungs from mice infected with PAO1, PAK, and KP. Data in (C), (D), and (G) are representative of data from three mice. Data in (E) and (F) are means ± SD from three mice (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01).

  • Fig. 2 MEG3-4 expression is regulated via a TLR4/NF-κB signaling pathway.

    (A) Primary alveolar macrophages (AMs) and AECII were infected with PAO1 at a multiplicity of infection (MOI) of 20:1 for 1 hour, polymyxin B (100 μg/ml) was added, and cells were cultured for another 1 hour to kill bacteria outside of the cell membrane. Samples were collected at multiple time points over 48 hours, and the expression of MEG3-4 in AMs and AECII cells is time-dependent, as detected by qRT-PCR. (B) Nuclear and cytosolic expression of MEG3-4 in primary AMs and AECII cells was detected by qRT-PCR. lncRNA Xist and H19 were used as nuclear and cytoplasmic controls, respectively. (C) TLR2 and TLR4 expression in AMs from wild-type (WT), Tlr2−/−, and Tlr4−/− mice was measured by immunoblotting. (D) WT, Tlr2−/−, and Tlr4−/− mice (n = 3) were infected with 5 × 106 CFU of PAO1 per mouse for 24 hours. AMs were collected to assess MEG3-4 expression. (E and F) MH-S cells were pretreated with indicated signaling pathway activators (a) or inhibitors (i) for 4 hours and then infected for 2 hours with PAO1 at an MOI of 20:1. MEG3-4 expression before and after infection was analyzed by qRT-PCR. (G and H) MH-S cells were transfected with control siRNA [scrambled siRNA (siNC)] and NF-κB p65 siRNA (si-p65) for 48 hours, respectively, and then infected with PAO1 at 20:1 MOI for 2 hours. Expression and phosphorylation of NF-κB p65 were measured by immunoblotting, and MEG3-4 transcripts were detected by qRT-PCR. Data in (C) and (G) are representative of three independent mice or cell samples. Data in (A), (B), (D) to (F), and (H) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01). NS, no significant change.

  • Fig. 3 MEG3-4 overexpression impairs host defense against P. aeruginosa.

    (A) Mice that received MEG3-4–overexpressing MH-S cells (MEG3-4 mice, each mouse preinfected with independently transfected cells with pWT-MEG3), mice that received EV-expressing cells, and control mice that did not receive cells (n = 6) were intranasally challenged with PAO1 at 5 × 106 CFU per mouse and observed up to 72 hours. Kaplan-Meier survival curves were obtained (P = 0.0217 between MEG3-4 and EV mice, log-rank test, n = 2 biological replicates). (B and C) Whole-animal imaging of bioluminescence was obtained using the IVIS XRII system at different time points after MEG3-4 and EV mice (n = 6) were intranasally challenged with PAO1 Xen-41 at 5 × 106 CFU per mouse (n = 2 biological replicates). (D and E) Bacterial burdens in the lungs and BALF were determined 24 hours after PAO1 infection. (F) PMN percentages were evaluated in BALF versus total nuclear cells using HEMA-3 staining. (G) Superoxides in AMs were detected by nitroblue tetrazolium (NBT) assay. RLU, relative units. (H) Mitochondrial potential of AMs was measured by JC-1 fluorescence assay. (I) Lung injury and inflammation were assessed by hematoxylin and eosin (H&E) staining of paraffin-embedded sections (black arrows indicate the region of insets with tissue injury and inflammatory influx). Scale bar, 100 μm. (J) Inflammatory cell infiltration was determined in the lungs in (I) (10 random areas from 3 triplicate samples). Data in (C) are means ± SD for two independent mouse lungs in (B). Data in (I) show one representative of three independent mice. Data in (J) are means ± SD for three independent mouse lungs in (I). Data in (D) to (H) are means ± SD for three independent mice (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01).

  • Fig. 4 MEG3-4 modulates IL-1β expression in mouse lungs.

    (A) Plasmids pWT-MEG3 and empty pcDNA3-EGFP were transfected into MH-S cells with Lipofectamine 2000 for 24 hours. MEG3-4–overexpressing and control MH-S cells were infected with PAO1 at 20:1 MOI for 30 min, and cell viabilities were determined by MTT. (B) Mice that received MEG3-4–overexpressing MH-S cells (MEG3-4 mice) and mice that received EV-expressing cells (n = 3) were infected with PAO1 at 5 × 106 CFU per mouse for 24 hours. Cytokine expression in BALF was assessed by ELISA. (C and D) Cytokines in mouse lungs were detected by qRT-PCR and immunoblotting. (E) Normal AMs were transfected with pWT-MEG3 and empty pcDNA3-EGFP (100 ng) for 24 hours. Cells were challenged with PAO1 at an MOI of 20:1 for 0, 30, 60, and 90 min. Confocal laser scanning microscopy showed the production of IL-1β in AMs using immunochemistry staining. Scale bar, 50 μm. (F and G) Indicated transcription factors in the lungs were determined by qRT-PCR and immunoblotting. (H) Inflammasome factors in the lungs were determined by immunoblotting. Data in (D), (E), (G), and (H) are representative of three independent mice or cell samples. Data in (A) to (C) and (F) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01).

  • Fig. 5 MEG3-4 binds miR-138 to compete the binding to IL-1β.

    (A) miRNA binding sites of MEG3-4 (www.microrna.org/). (B) Mice (n = 3) were intranasally infected with 5 × 106 CFU PAO1 for 24 hours, and the lungs were lysed to assess miR-129-5p, miR-138, and miR-136. (C) In situ analysis with a digoxigenin (DIG)–labeled miR-138 probe (brown) in PAO1-infected and control lungs. Nuclei were stained by hematoxylin (blue). Scale bar, 100 μm. (D) Northern blots of miR-138 in lungs after PAO1, PAK, and KP infection. (E) Duplex formations between MEG3-4 (bottom) and miR-138 (middle). Target mutagenesis sites (top) are indicated. (F) MEG3-4 WT and mutant cloned to downstream of the luciferase coding region in pGL3 and cotransfected in MH-S with 138-m. Luciferase activities were determined using Luciferase Reporter Assay. (G) IL-1β 3′UTRs contain miR-138 binding sequence. Predicted duplex formations between IL-1β 3′UTR (bottom) and miR-138 (middle). (H) IL-1β 3′UTR and its mutant cloned to pGL3 and cotransfected into MH-S cells with 138-m. Luciferase activities were determined by Luciferase Reporter Assay. (I) IL-1β 3′UTR and its mutant cloned to pGL3 and cotransfected into MH-S cells with 138-m and pWT-MEG3 or with 138-m and pMU-MEG3 [mutant (mu)]. Luciferase activities were determined by Luciferase Reporter Assay. Empty pcDNA3-EGFP vector was used as a control. (J) Binding of Ago2 with MEG3-4, miR-138, and IL-1β mRNA was detected by individual cross-linking immunoprecipitation (iCLIP) using anti-Ago2 antibody and qRT-PCR. (K) Plasmids pWT-MEG3 and empty pcDNA3-EGFP transfected into MH-S. Binding of miR-138 to MEG3-4 or IL-1β mRNA in MEG3-4–overexpressing and EV cells was detected by LAMP assay and endpoint gel PCR. miRNA alone without DIG labeling was used as a negative control, whereas these transcripts in total MH-S cell extracts (input) were used as a positive control. In (J) and (K), binding of miR-320b and IL-1 receptor–associated kinase 4 (IRAK4) was used as a positive control. In (K), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a negative control, and 18S and 28S rRNAs were used as loading controls. Data in (C), (D), (J), and (K) show one representative of three independent mice or cell samples. Data in (B), (F), and (H) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01). IP, immunoprecipitation.

  • Fig. 6 MEG3-4 modulates IL-1β and subsequent inflammatory responses by tightly modulating miR-138 expression.

    (A) MH-S cells were infected with PAO1 at 20:1 for 2 hours. Copies of IL-1β mRNA, miR-138, and MEG3-4 in each MH-S cell were detected by absolute qRT-PCR. (B) Normal AMs (n = 3) transfected with IL-1β CRISPR-Cas activation plasmids or control plasmids (50 ng) or for 48 hours. TNF-α, IL-1β, and IL-6 were detected by qRT-PCR. (C and D) MEG3-4 and miR-138 in transfected AMs were measured by qRT-PCR. (E) Mice were intravenously injected with vehicle containing either NS-m or 138-m (50 μg per mouse) 24 and 48 hours (two time points in different animals) before PAO1 challenge. Kaplan-Meier survival curves of PAO1-infected NS-m– or 138-m–injected mice (n = 6, two independent experiments). Survival was determined up to 96 hours (P = 0.0177, log-rank test). (F) 138-m– and NS-m–injected mice (n = 3) were infected with PAO1 at 5 × 106 CFU per mouse for 24 hours. Expression of cytokines in mouse lungs was detected by immunoblotting. (G) qRT-PCR analysis of MEG3-4 expression in the lung from PAO1-infected NS-m– and 138-m–injected mice. (H) Proposed model for the role of lncRNA MEG3-4 in regulating IL-1β expression by competitively binding miR-138. Data in (A) to (D) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01). Error bars represent SD. Data in (F) and (G) are representative of three independent mice or cell samples.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/536/eaao2387/DC1

    Fig. S1. Characterization of lncRNA MEG3.

    Fig. S2. MEG3-4 inhibition signaling in alveolar macrophage cells is TLR4-specific during S. aureus infection.

    Fig. S3. Immunoblotting validation of signaling factors in inhibitor- or activator-treated MH-S cells.

    Fig. S4. Dissection of signaling molecules in PAO1-infected MH-S cells.

    Fig. S5. Densitometric quantification of the immunoblotting data.

    Fig. S6. Restoration of the phenotype in a MEG3-4–overexpressing model.

    Fig. S7. Imaging of pyroptosis in MH-S cells.

    Fig. S8. Functional analysis of miRNAs generated by MEG3-4.

    Fig. S9. miR-138 regulates IL-1β expression and cell viability in alveolar macrophages.

    Fig. S10. miR-138 enhances host defense against P. aeruginosa by repressing IL-1β expression in mouse lungs.

    Fig. S11. MEG3-4 overexpression phenotypes in mice were reversed by treatment with 138-m.

    Fig. S12. Analysis of MEG3 function and expression in human alveolar macrophages.

    Fig. S13. MEG3-4 overexpression inhibits p53 expression in mouse B16 melanoma tumor cells.

    Table S1. lncRNA expression in response to PAO1 infection.

    Table S2. Primers used in this study.

  • Supplementary Materials for:

    MEG3-4 is a miRNA decoy that regulates IL-1β abundance to initiate and then limit inflammation to prevent sepsis during lung infection

    Rongpeng Li, Lizhu Fang, Qinqin Pu, Huimin Bu, Pengcheng Zhu, Zihan Chen, Min Yu, Xuefeng Li, Timothy Weiland, Arvind Bansal, Shui Qing Ye, Yuquan Wei, Jianxin Jiang,* Min Wu*

    *Corresponding author. Email: min.wu{at}med.und.edu (M.W.); hellojjx{at}126.com (J.J.)

    This PDF file includes:

    • Fig. S1. Characterization of lncRNA MEG3.
    • Fig. S2. MEG3-4 inhibition signaling in alveolar macrophage cells is TLR4-specific during S. aureus infection.
    • Fig. S3. Immunoblotting validation of signaling factors in inhibitor- or activator-treated MH-S cells.
    • Fig. S4. Dissection of signaling molecules in PAO1-infected MH-S cells.
    • Fig. S5. Densitometric quantification of the immunoblotting data.
    • Fig. S6. Restoration of the phenotype in a MEG3-4–overexpressing model.
    • Fig. S7. Imaging of pyroptosis in MH-S cells.
    • Fig. S8. Functional analysis of miRNAs generated by MEG3-4.
    • Fig. S9. miR-138 regulates IL-1β expression and cell viability in alveolar macrophages.
    • Fig. S10. miR-138 enhances host defense against P. aeruginosa by repressing IL-1β expression in mouse lungs.
    • Fig. S11. MEG3-4 overexpression phenotypes in mice were reversed by treatment with 138-m.
    • Fig. S12. Analysis of MEG3 function and expression in human alveolar macrophages.
    • Fig. S13. MEG3-4 overexpression inhibits p53 expression in mouse B16 melanoma tumor cells.
    • Table S1. lncRNA expression in response to PAO1 infection.
    • Table S2. Primers used in this study.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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