Journal: Science signaling

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

doi: 10.1126/scisignal.aao2387

Figure Lengend Snippet: (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.

Article Snippet: All of the nucleases, polymerases, and ligases used in molecular cloning were bought from New England Biolabs Inc. Transfection of siRNA, plasmid, activators, and inhibitors NF-κB p65 (sc-29411), TLR2 (sc-40257), TLR4 (sc-40261), siNCs (sc-37007), IL-1β CRISPR-Cas9 activation (sc-421097-ACT), and control plasmids (sc-437275) were obtained from Santa Cruz Biotechnology.

Techniques: Binding Assay, Infection, In Situ, Labeling, Staining, Northern Blot, Mutagenesis, Clone Assay, Luciferase, Reporter Assay, Sequencing, Plasmid Preparation, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Transfection, Lamp Assay, Negative Control, Positive Control, Immunoprecipitation

Journal: Science signaling

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

doi: 10.1126/scisignal.aao2387

Figure Lengend Snippet: (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).

Article Snippet: All of the nucleases, polymerases, and ligases used in molecular cloning were bought from New England Biolabs Inc. Transfection of siRNA, plasmid, activators, and inhibitors NF-κB p65 (sc-29411), TLR2 (sc-40257), TLR4 (sc-40261), siNCs (sc-37007), IL-1β CRISPR-Cas9 activation (sc-421097-ACT), and control plasmids (sc-437275) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Confocal Laser Scanning Microscopy, Staining

Journal: Science signaling

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

doi: 10.1126/scisignal.aao2387

Figure Lengend Snippet: (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.

Article Snippet: All of the nucleases, polymerases, and ligases used in molecular cloning were bought from New England Biolabs Inc. Transfection of siRNA, plasmid, activators, and inhibitors NF-κB p65 (sc-29411), TLR2 (sc-40257), TLR4 (sc-40261), siNCs (sc-37007), IL-1β CRISPR-Cas9 activation (sc-421097-ACT), and control plasmids (sc-437275) were obtained from Santa Cruz Biotechnology.

Techniques: Infection, Quantitative RT-PCR, Transfection, CRISPR, Activation Assay, Injection, Expressing, Western Blot, Binding Assay