Journal: MedComm
Article Title: Long Noncoding RNA Interleukin 6 Antisense RNA 1 Promotes Inflammatory Effects in Lung Macrophages via Exosomes Through the S100A9/TLR4 Pathway in Chronic Obstructive Pulmonary Disease Progression
doi: 10.1002/mco2.70204
Figure Lengend Snippet: IL6‐AS1 exhibits specific binding to S100A9 in both in vivo and in vitro settings. (A) Diagrammatic representation illustrates RNA‐seq conducted on HFL1 cells following overexpression of IL6‐AS1 . (B) Metascape enrichment results for differentially upregulated genes in IL6‐AS1 mice in the air group, IL6‐AS1 mice in the smoke group, and genes upregulated following IL6‐AS1 overexpression in HFL cells. (C) qRT‐PCR for the expression of inflammation‐associated genes (IL‐6, IL‐8, ICAM‐1, CCL‐2/7, and CXCL‐3/10) in HFL1 cells after the IL6‐AS1 overexpression. ( n = 4 biological replicates). (D) ChIRP assays were conducted using either an unspecific lacZ probe or probes designed for IL6‐AS1 in human lung tissue, followed by silver staining and mass spectrometric sequencing. (E) A PPI network was constructed by combining the distinct binding proteins identified in the mass spectrometry results of the target group with the differentially expressed COPD‐related genes identified in the RNA‐seq. (F) Western blot analysis of the proteins from the proteomics screen after ChIRP assays shows the specific interaction of IL6‐AS1 with S100A9 protein in lung homogenates. (G) RIP‐qPCR analysis with anti‐S100A9 antibody in wild‐type (NC) and IL6‐AS1 mice. Anti‐IgG antibody acted as a negative control ( n = 5). (H) RNA immunoprecipitation followed by quantitative PCR (RIP‐qPCR) analysis using anti‐S100A9 antibody in HFL1 cells. Anti‐IgG antibody and U1 served as negative controls. ( n = 3 biological replicates). (I) RIP‐qPCR analysis using anti‐S100A9 antibody in HFL1 cells after stimulation with recombinant S100A9 protein (rp‐S100A9). Anti‐IgG antibody served as a negative control. ( n = 3 biological replicates). (J) Lung tissue sections from the four groups of mice were subjected to IHC staining for S100A9. ( n = 7 for Smoke+ IL6‐AS1, n = 5 for other three groups). (K) The concentration of S100A9 protein in the lung tissue homogenates and serum of the four groups of mice was determined by ELISA. ( n = 7 for Smoke+ IL6‐AS1, n = 5 for other three groups). (L) Western blot illustration reveals the presence of phosphorylated p65 and p38 following stimulation with rp‐S100A9 for 0, 1, 6, 12, and 24 h ( n = 3 biological replicates). (M) qRT‐PCR evaluation of the expression of CCL‐2 and IL‐6 in IL6‐AS1 ‐augmented HFL1 cells following stimulation with rp‐S100A9 at the 1 and 24‐h time points. ( n = 4 biological replicates). Data are presented as mean ± SD. p Values in charts were determined by one‐way ANOVA Bonferroni's multiple comparisons test (G, H, and I), one‐way ANOVA Tukey's multiple comparisons test (C, L, and M), and unpaired two‐tailed Student's t ‐test (J and K).
Article Snippet: Human lung fibroblast 1 (HFL1) cells were sourced from ATCC (CCL‐153; Manassas, USA), which was cultured in F‐12K medium (Kaighn's modification of Ham's F‐12) supplemented with 10% FBS (Gibco) and 1% P/S (Invitrogen).
Techniques: Binding Assay, In Vivo, In Vitro, RNA Sequencing, Over Expression, Quantitative RT-PCR, Expressing, Silver Staining, Sequencing, Construct, Mass Spectrometry, Western Blot, Negative Control, RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Recombinant, Immunohistochemistry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test
![IL6‐AS1 on fibroblasts can facilitate the binding of S100A9 protein to TLR4 and AGER receptors on macrophages. (A) Schematic representation of the coculture system involving macrophages and fibroblasts. (B) qRT‐PCR assay determining CD86 and CD11b expression in THP‐1 cells following coculture, after stimulation with PMA solely or in conjunction with rp‐S100A9 ( n = 4 biological replicates). (C) qRT‐PCR assay measuring the expression of IL6‐AS1 in HFL1 and Thp‐1 cells following coculture. ( n = 4 biological replicates). (D) qRT‐PCR assay analyzing the expression of inflammation‐associated genes ( IL‐6 , IL‐8 , CCL‐2/7 , and CXCL‐3/10 ) in THP‐1 cells after coculture ( n = 4 biological replicates). (E) qRT‐PCR assay of IL‐10 , CD206 , CD86 , and TNF‐α expression in Thp‐1 cells following coculture ( n = 4 biological replicates). (F) Co‐IP analysis of Thp‐1 cocultured with IL6‐AS1 ‐overexpressed HFL1 using anti‐S100A9 antibody. Western blot was used to verify the co‐IP results with anti‐S100A9, anti‐TLR4, and anti‐AGER antibodies ( n = 3 biological replicates). (G and H) IF double staining was performed using anti‐S100A9/anti‐TLR4 antibody (G) or anti‐S100A9/anti‐AGER antibody (H) in mouse lung tissue sections extracted from 4 distinct groups of mice. ( n = 4). (I) Western blot revealing the presence of TLR4 and AGER in four distinct mouse groupings, utilizing Actin as a control reference. ( n = 5, n = 7 [Smoke+ IL6‐AS1 ]). Data shown mean ± SD. p Values shown in charts are determined by multiple two‐tailed Students’ t ‐tests (B, C, D, E, and F) and one‐way ANOVA Bonferroni's multiple comparisons test (G and I).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1923/pmc12141923/pmc12141923__MCO2-6-e70204-g005.jpg)
