Figure 6: The transcriptional programme driven by PBX3 in HCC TICs.

(a) Piediagram summarizing the genomic occupancy of PBX3-bound regions as revealed by ChIP-seq. (b) Line diagram showing the distances of PBX3-bound peaks to the TSS. (c) Venn diagram illustrating direct target genes regulated by PBX3. (d) Gene Ontology (GO) term analysis of differentially regulated genes, as revealed using RNA-seq, in PBX3-overexpressed SMMC7721 cells over vector control. (e) qRT–PCR results showing α2δ1 mRNA levels in PBX3-overexpressed α2δ1⁻Huh7 cells and PBX3 knockdown Hep-12 cells. (f,g) Western blot results demonstrating the change of endogenous α2δ1 proteins following forced expression of PBX3 in α2δ1⁻Huh7 cells or shRNA-mediated knockdown of PBX3 in Hep-12 cells, respectively. (h) Schematic representation of the CACNA2D1 promoter that is fused to a luciferase reporter gene. The WT and mutant (Mut) sequences of the consensus potential PBX3-binding site used to make CACNA2D1 WT and Mut reporters, respectively, are indicated. (i,j) PBX3 activates the activity of the WT CACNA2D1 promoter in a dose-dependent manner (i), but not that of the mutant-type promoter containing mutant consensus potential PBX3-binding site (j). Luciferase reporter assay was performed in Hep-11 cells by co-transfection CACNA2D1 reporter with either pcDNA3-PBX3 or empty vector. Co-transfection of PRL-TK served as an internal control. Luciferase values were normalized to Renilla reporter activity. Data are presented as mean±s.d. of three independent experiments with triplicates. *Student’s t-test. (k) EMSA analysis was performed by incubating biotin-labeled oligonucleotide target containing consensus potential PBX3-binding site with Hep-11 or Hep-12 cell lysates. (l) ChIP assay was performed with control IgG or anti-PBX3 antibody in Hep-12 cells. The precipitated DNAs were amplified with primer pairs specific for flank sequence of the PBX3-binding sites in the CACNA2D1 promoter using PCR. (m) The correlation between the expression of PBX3 and its direct target CACNA2D1 in HCC tissues as detected using qRT–PCR. (n) Western blot results showing the correlation between PBX3 and α2δ1 in 16 pairs of HCC (T) and adjacent normal tissues (N).