Journal: Molecular Therapy. Nucleic Acids
Article Title: MiR-449a antagonizes EMT through IL-6-mediated trans -signaling in laryngeal squamous cancer
doi: 10.1016/j.omtn.2024.102140
Figure Lengend Snippet: miR-449a directly targets IL-6R and switches off IL-6 trans -signaling (A) The putative miR-449a binding sites in IL-6R were predicted using online bioinformatics tools. The interaction between miR-449a and IL-6R was verified using a dual luciferase reporter assay. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001 Student’s t test vs. control group. Each experiment was repeated three times. Bars indicate SDs. (B) RIP assay of the enrichment of Ago2 on IL-6R and miR-449a transcripts normalized to a negative control irrelevant IgG antibody (CTR) in HNO-210 cell line overexpressing miR-449a. (C) Levels of sIL-6R and IL-6R in HNO-210 cell culture medium by quantitative ELISA (left and right) and expression levels of sIL-6R in HNO-210 cells by qRT-PCR (last panel). The experiments were repeated three times in triplicate. Bars, SDs. (D) Western blots analysis of transduced HNO-210 cell models in different experimental conditions: (i) 0.1% serum starvation, (ii) Hyper IL-6 stimulation (20 ng/mL for 30 min), (iii) sgp130fc treatment (1,000 ng/mL for 1 h), and sgp130fc pretreatment followed by Hyper IL-6 stimulation. miR-449a modulation of IL-6 trans -signaling mediated by pStat3 Tyr705 – pERK (Thr202; Tyr204)—pAKT Ser473. Vinculin was used as loading control. The experiments were repeated at least three times giving always similar results. Columns represent the intensity of the different bands evaluated as arbitrary units. Bars are SDs. ANOVA ∗p < 0.01; ∗∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001. (E) Relative expression levels of SNAI2, E-cadherin, and N-cadherin of transduced HNO-210 cell models in different experimental conditions: (i) 0.1% serum starvation, (ii) Hyper IL-6 stimulation (20 ng/mL for 30 min), (iii) sgp130fc treatment (1,000 ng/mL for 1 h) and sgp130fc pretreatment followed by Hyper IL-6 stimulation. ANOVA ∗<0.01 ∗∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001. All data are representative of three independent experiments.
Article Snippet: Cells was analyzed in different experimental conditions: 0.1% Serum Starvation, Hyper IL-6 R&D Systems (Recombinant Human IL-6/IL-6R Complex Chimera - Cat# 8954-SR) stimulation (20 ng/mL for 30 min), sgp130fc (Recombinant Human gp130 Fc Chimera - Cat# 671-GP) treatment (1,000 ng/mL for 1 h), sgp130fc pretreatment followed by Hyper IL-6 stimulation.
Techniques: Binding Assay, Luciferase, Reporter Assay, Negative Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot
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