Fig. 4

YY1 promotes HCC tumorigenesis via induction of EGFR transcription activity under conditions of CRNDE overexpression. A, B Western blot and qRT-PCR analysis of YY1 and EGFR protein and EGFR mRNA expression in Huh7 cells under conditions of CRNDE overexpression and silencing of YY1, respectively. C Relative luciferase activities of the EGFR promoter in Huh7 cells with CRNDE overexpression and shRNA-silenced YY1 were determined using the reporter assay. D Schematic representation of YY1 binding sites in the 0.6 kb EGFR promoter region. Partial wild-type (WT) and mutant YY1 binding sequences are listed below, including mutant YY1 at binding site 1 (M1), mutant YY1 at binding site 2 (M2), and double mutant YY1 at binding sites 1 and 2 (M3). E Reporter assay of the relative luciferase activity of each promoter in Huh7 cells transfected with pcDNA3-control or CRNDE overexpression constructs. Luciferase activities of the EGFR promoter with mutant YY1 binding site 1 (M1), YY1 binding site 2 (M2), and YY1 binding site 1 and 2 (M3), taking wild-type (WT) EGFR promoter activity as 100%. F Schematic diagram of the promoter and other regions of the EGFR gene. The YY1 binding site and transcriptional initiation site (+ 1) are indicated. YY1 binding to the promoter region of EGFR, ranging from 464 to 476 bp upstream of the transcription start site, and a 249-bp PCR product containing the YY1-binding site on the EGFR promoter are shown. The positions of PCR primers and elements relative to the transcription initiation site are numbered. G–J YY1 binding to the EGFR promoter in Hep3B (control and CRNDE depletion) and Huh7 (pcDNA3 and CRNDE overexpression) cells was determined via ChIP PCR or ChIP-q-PCR. Images of representative gels of ChIP PCR products. The chromatin of cells subjected to the indicated treatments was immunoprecipitated with YY1 antibody or IgG control. Co-IP DNA for the YY1 binding region (EGFR) or negative control region (GAPDH) was detected using PCR. Statistical significance (p value) was calculated with the two-tailed Student's t-test for a single comparison between two groups. Data are presented as mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001)