Fig 2: The expression pattern and correlations of LINC01578/NFKBIB/YY1 in colon cancer liver metastasis. (A) NFKBIB expression in 60 colon cancer tissues without metastasis and 10 colon cancer tissues with metastasis was measured by qRT-PCR. P = 0.0056 by the Mann–Whitney test. (B) The correlation between NFKBIB and LINC01578 expression in these 70 colon cancer tissues. P < 0.0001, R = -0.4878 by Pearson's correlation analysis. (C) The correlation between NFKBIB and LINC01578 expression in GSE17538 dataset. P < 0.0001, R = -0.4402 by Pearson's correlation analysis. (D) YY1 expression in 60 colon cancer tissues without metastasis and 10 colon cancer tissues with metastasis was measured by qRT-PCR. P = 0.0090 by the Mann–Whitney test. (E) The correlation between YY1 and LINC01578 expression in these 70 colon cancer tissues. P = 0.0004, R = 0.4150 by the Pearson correlation analysis. (F) The correlation between YY1 and LINC01578 expression in GSE17538 dataset. P < 0.0001, R = 0.3784 by the Pearson correlation analysis.

Fig 3: LINC01578 is upregulated by NF-?B and YY1. (A) The predicted NF-?B and YY1 binding sites on LINC01578 promoter. NF-?B and YY1 bind sites were at -438 and -4 positions relative to the transcription start site, respectively. (B) ChIP assays were performed in DLD-1 cells to measure the binding of NF-?B and YY1 on LINC01578 promoter. A distant region without NF-?B and YY1 binding sites was used as NC (P3). (C) Luciferase reporter assays for DLD-1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with PBS or 10 ng·mL-1 TNF-a for 24 h. Nuclear p65 levels of DLD-1 cell treatment with PBS or 10 ng·mL-1 TNF-a for 24 h were detected by western blot. (D) Luciferase reporter assays for DLD-1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with DMSO or 5 µm JSH-23 for 24 h. Nuclear p65 levels of DLD-1 cell treatment with DMSO or 5 µm JSH-23 for 24 h were detected by western blot. (E) Luciferase reporter assays for DLD-1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and p65 overexpression vector. p65 overexpression efficiencies were detected by western blot. (F) Luciferase reporter assays for DLD-1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against p65. p65 knockdown efficiencies were detected by western blot. (G) Luciferase reporter assays for DLD-1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and YY1 overexpression vector. YY1 overexpression efficiencies were detected by western blot. (H) Luciferase reporter assays for DLD-1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against YY1. YY1 knockdown efficiencies were detected by western blot. (I) LINC01578 expression in DLD-1 cells treated with PBS or 10 ng·mL-1 TNF-a for 24 h. (J) LINC01578 expression in DLD-1 cells treated with DMSO or 5 µm JSH-23 for 24 h. (K) LINC01578 expression in DLD-1 cells transfected with p65 overexpression vector. (L) LINC01578 expression in DLD-1 cells transfected with siRNAs against p65. (M) LINC01578 expression in DLD-1 cells transfected with YY1 overexpression vector. (N) LINC01578 expression in DLD-1 cells transfected with siRNAs against YY1. Data are shown as mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, by one-way ANOVA followed by Dunnett's multiple comparisons test (B) or Student's t-test (C-N).