HBP1-mediated transcriptional repression of AFP inhibits hepatoma progression

Fig. 1. HBP1 regulates AFP expression. a There is a negative correlation between the expression of HBP1 and AFP in hepatoma tissues. The gene expression data were derived from the primary tumors who received surgery or TACE and then monitored for relapse for up 10 years. The protein levels of HBP1 and AFP were measured by IHC in hepatoma tissues. Scale bar, 100 μm. b Elevated expression of HBP1 in tumor is associated with higher survival rate of hepatoma patients. The overall survival curves were performed in two groups of hepatoma patients with high or low level of HBP1. The protein levels of HBP1 were analyzed based on tumor TNM classification. c There is a negative correlation between the expression of HBP1 and HBV infection. The protein level of HBP1 was measured by IHC in hepatoma tissues. d HBP1 overexpression decreases AFP protein and mRNA expression. The protein levels of HBP1 and AFP in cell lysate and supernatant were measured by Western blotting in HepG2 and PLC5 cells transfected with pCDNA3-HBP1 or pCDNA3 (as a control). GAPDH and GST were used as a loading control respectively (left panel). The mRNA level of AFP was measured by Realtime-PCR in HepG2 and PLC5 cells…

Fig. 2. HBP1 represses the AFP gene through binding an affinity site in the AFP promoter. a Relative activity of HBP1 on the AFP promoters with various lengths. b HBP1 suppresses AFP promoter activity in a dose-dependent manner. Luciferase activity was detected in 293 T cells co-transfected with the indicated reporter genes and HBP1 plasmids. c The integrity of affinity site is indispensable for HBP1 suppressing AFP promoter in vivo. Shown is schematic diagram of the wild type AFP promoter and its mutant promoter (left panel) and the relative activities of HBP1 on the wild type AFP promoter and mutant AFP promoters (right panel). d Relative activities of HBP1 and associated mutants on the AFP promoter in co-transfected 293 T cells. Shown is schematic diagram of wild-type HBP1 and associated mutants (left panel). Luciferase activity was determined after transfection (right panel). e Expression of exogenous HBP1 decreases AFP protein level. Two hundred-ninety three T cells were transfected with HBP1 and associated mutants. The protein level was measured by Western blotting. f HBP1 occupies its affinity site in the AFP promoter. EMSA assays were performed by using a biotin-labeled…

Fig. 3. HBP1 acetylation at K419 is required for HBP1 suppression of AFP transcription. a Schematic diagram of wild-type HBP1 and associated point mutants. b The acetylation of HBP1 at K419 is indispensable for its suppression of AFP promoter. Two hundred-ninety three T cells were co-transfected with AFP promoter and HBP1 or mutant expression plasmids. Luciferase activity was determined after transfection. c The acetylation of HBP1 at K419 is indispensable for its suppression of AFP mRNA. The mRNA level of AFP was measured by Realtime-PCR in HepG2 cells transfected with HBP1 or associated mutant vectors individually. d The acetylation of HBP1 at K419 is indispensable for its suppression of AFP protein. HepG2 cells were transfected with HBP1 or associated mutant vectors. The protein levels of HBP1 and AFP were measured by Western blotting. Error bars represent S.D. **, p < 0.01

Fig. 4. HBx attenuates the suppression of HBP1 on AFP through inhibiting binding of HBP1 to the AFP promoter. a HBx rescues the suppression of HBP1 on AFP protein. HepG2 cells were co-transfected HBP1 with or without HBx expression plasmid. The protein levels of HBP1, AFP and HBx were measured by Western blotting. b HBx rescues the suppression of HBP1 on AFP mRNA. The mRNA level of AFP was measured by Realtime-PCR in HepG2 cells transfected with HBP1 with or without HBx expression plasmid. c HBx rescues the suppression of HBP1 on AFP promoter. Two hundred-ninety three T cells were co-transfected with AFP promoter and HBP1 with or without HBx expression plasmid. Luciferase activity was determined after transfection. d HBx inhibits the interaction between HBP1 and AFP promoter. Two hundred-ninety three T cells were transfected with HA-HBP1 with or without HBx expression plasmid. The region from position − 1600 to position − 1448 contains the HBP1 affinity site and was analyzed by specific PCR. e HBP1 interacts with HBx. Two hundred-ninety three T cells were co-transfected with HA-HBP1 and FLAG-HBx. IP assay was carried out by using anti-FLAG/HA antibody and followed by Western blotting…

Fig. 5. Icaritin promotes the suppression of HBP1 on AFP through enhancing HBP1 binding to AFP promoter. a Icaritin promotes the suppression of HBP1 on AFP protein. HepG2 cells were transfected HBP1 with or without Icaritin treatment. The protein levels of HBP1 and AFP were measured by Western blotting. b Icaritin promotes the suppression of HBP1 on AFP mRNA. The mRNA level of AFP was measured by Realtime-PCR in HepG2 cells transfected HBP1 with or without Icaritin treatment. c Icaritin promotes the suppression of HBP1 on AFP promoter. Two hundred-ninety three T cells were co-transfected with AFP promoter and HBP1 with or without Icaritin treatment. Luciferase activity was determined after transfection. d Icaritin promotes the interaction between HBP1 and AFP promoter. Two hundred-ninety three T cells were transfected HA-HBP1 with or without Icaritin treatment. The region from position − 1600 to position − 1448 contains the HBP1 affinity site and was analyzed by specific PCR. e HBP1 protein level is elevated in the presence of Icaritin. HepG2 cells were treated with Icaritin. The protein level of HBP1 was measured by Western blotting (top panel). The mRNA level of HBP1 was measured by…

Fig. 6. HBP1 inhibits the functions of PTEN and caspase-3 through suppressing AFP in hepatoma cells. a AFP rescues the effect of HBP1 on PTEN, caspase-3, and MMP9 protein levels. HepG2 cells were co-transfected HBP1 with or without AFP expression plasmid. The protein levels of HBP1, AFP, PTEN, phospho-AKT, AKT, pro-caspase-3, cleaved-caspase-3 and MMP9 were measured by Western blotting. b AFPshRNA rescues the effect of HBP1shRNA on PTEN, caspase-3, and MMP9 protein levels. HepG2 cells were co-transfected HBP1shRNA with or without AFPshRNA expression plasmid. The protein levels of HBP1, AFP, PTEN, phospho-AKT, AKT, pro-caspase-3, cleaved-caspase-3 and MMP9 were measured by Western blotting. c HBx rescues the effect of HBP1 on PTEN, caspase-3, and MMP9 protein levels. HepG2 cells were co-transfected HBP1 with or without HBx expression plasmid. The protein levels of HBP1, AFP, PTEN, phospho-AKT, AKT, pro-caspase-3, cleaved-caspase-3 and MMP9 were measured by Western blotting. d Icaritin enhances the effect of HBP1 on PTEN, caspase-3, and MMP9 protein levels. HepG2 cells transfected with HBP1 were treated with or without Icaritin. The protein levels of HBP1, AFP, PTEN, phospho-AKT, AKT,…

Fig. 7. (See Fig. 7 continuation on next page.)

Fig. 8. HBP1 deletion aggravates DEN/CCl4-induced hepatoma. a Representative images of hepatoma sections from 20 weeks after DEN/CCl4 treatment in wild type or HBP1−/− mice. b The protein levels of HBP1 and AFP in the hepatoma described in Fig. 8a were measured by Western blotting. β-actin was used as a loading control. c The mRNA level of AFP was measured by Realtime-PCR in the hepatoma described in Fig. 8a. d Serum AFP level in the mice described in Fig. 8a. e The IHC analyses of Ki67 in the mice described in Fig. 8a. f Model of the role of HBP1 in inhibiting the occurrence and development of hepatoma by repressing the expression of AFP. Scale bar, 100 μm. Bar graphs show numbers of Ki67-positive cells per 100 × field. Error bars represent S.D. *, p < 0.05, **, p < 0.01