FIGURE 5.

IL6‐AS1 interacts with EBF1 to regulate IL‐6 expression by affecting the binding of EBF1 to the IL‐6 promoter. (A) RNA pull‐down assay using IL6‐AS1 sense and antisense RNAs in HBF cells, followed by silver staining. Black arrow indicates EBF1. The interaction between IL6‐AS1 and EBF1 was confirmed by Western blotting with the extract of the RNA pull‐down assay. ILF2 and GPR94 were also detected. (B) RIP‐qPCR analysis using an anti‐EBF1 antibody showed that IL6‐AS1 interacts with endogenous EBF1 in HBF and HFL1 cells. U1 was used as the negative control (two‐way ANOVA, n = 3 biological replicates). (C) RIP‐qPCR analysis with an anti‐EBF1 antibody was conducted after overexpression of IL6‐AS1 in HBF and HFL1 cells (two‐way ANOVA, n = 3 biological replicates). (D) Schematic representation of potential EBF1 binding sites on the IL‐6 promoter. As promoters considered to be located 2000 bp upstream of the transcription start site (TSS), we designed six pairs of chromatin immunoprecipitation (ChIP)‐qPCR primers to cover the IL‐6 promoter region, with the fifth and sixth primers containing two EBF1 binding sites. (E) ChIP‐PCR analysis of EBF1 occupancy on the IL‐6 promoter in HBF and HLF cells. IgG was used as the negative control (two‐way ANOVA, n = 3 biological replicates). (F) ChIP‐PCR analysis of EBF1 occupancy on the IL‐6 promoter after transfection with an IL6‐AS1 silencer (SSIL6‐AS1) in HBF and HLF cells (two‐way ANOVA, n = 3 biological replicates). (G) Luciferase activity in the IL‐6 promoter following cotransfection of the IL6‐AS1 silencer (SSIL6‐AS1) or IL6‐AS1 overexpression vector (two‐way ANOVA, n = 4 biological replicates). (H) Luciferase activity in the IL‐6 promoter following cotransfection of an EBF1 small interfering RNA (siRNA) (siEBF1‐1) and EBF1 overexpression vector (two‐way ANOVA, n = 4 biological replicates). (I) Schematic representation of the two mutation sequences of potential EBF1 binding sites on the IL‐6 promoter. (J) Luciferase activity in the IL‐6 promoter following transfection with a reporter containing wild‐type or mutant IL‐6 promoter (one‐way ANOVA, n = 4 biological replicates). (K and L) qRT‐PCR and ELISA analysis of IL‐6 expression after transfection with two EBF1 siRNAs in HBF cells (K) or HLF cells (L) (one‐way ANOVA, n = 4 biological replicates). (M and N) qRT‐PCR and ELISA analysis of IL‐6 expression after overexpression of EBF1 in HBF cells (M) or HLF cells (N) (one‐way ANOVA, n = 4 biological replicates). (O and P) Expression of IL‐6 in HBF cells following cotransfection with IL6‐AS1 overexpression vector and EBF1 siRNA (SiEBF1‐1), determined by qRT‐PCR (O) and ELISA (P) (one‐way ANOVA, n = 5 biological replicates). (Q and R) Expression of IL‐6 in HBF cells following cotransfection with an IL6‐AS1 Smart Silencer (SSIL6‐AS1) and EBF1 overexpression vector, determined by qRT‐PCR (Q) and ELISA (R) (one‐way ANOVA, n = 5 biological replicates). Error bars represent means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001