Hepatitis B virus X protein accelerates hepatocarcinogenesis with partner survivin through modulating miR-520b and HBXIP

Abstract

Background: Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. However, the underlying mechanism remains elusive. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The oncopreotein hepatitis B X-interacting protein (HBXIP), a target of miR-520b, is involved in the development of cancer. In this study, we focus on the investigation of hepatocarcinogenesis mediated by HBx.

Methods: The expression of HBx and survivin was examined in the liver tissues of HBx-Tg mice. The effect of HBx/survivin on the growth of LO2-X-S cells was determined by colony formation and transplantation in nude mice. The effect of HBx/survivin on promoter of miR-520b was determined by Western blot analysis, luciferase reporter gene assay, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP), respectively. The expression of HBx, survivin and HBXIP was detected by immunohistochemistry and real-time PCR in clinical HCC tissues, respectively. The DNA demethylation of HBXIP promoter was examined. The functional influence of miR-520b and HBXIP on proliferation of hepatoma cells was analyzed by MTT, colony formation, EdU and transplantation in nude mice in vitro and in vivo.

Results: In this study, we provided evidence that HBx up-regulated survivin in the liver cancer tissues of HBx-Tg mice aged 18 M. The engineered LO2 cell lines with survivin and/or HBx were successfully established, termed LO2-X-S. MiR-520b was down-regulated in LO2-X-S cells and clinical HCC tissues. Our data revealed that HBx survivin-dependently down-regulated miR-520b through interacting with Sp1 in the cells. HBXIP was highly expressed in LO2-X-S cells, liver cancer tissues of HBx-Tg mice aged 18 M and clinical HCC tissues (75.17%, 112/149). The expression level of HBXIP was positively associated with those of HBx or survivin in clinical HCC tissues. In addition, we showed that HBx survivin-dependently up-regulated HBXIP through inducing demethylation of HBXIP promoter in LO2-X-S cells and clinical HCC tissues. In function, low level miR-520b and high level HBXIP mediated by HBx with partner survivin contributed to the growth of LO2-X-S cells in vitro and in vivo.

Conclusion: HBx accelerates hepatocarcinogenesis with partner survivin through modulating tumor suppressor miR-520b and oncoprotein HBXIP.

Figure 1

HBx accelerates hepatocarcinogenesis with partner survivin. (A) The expression of survivin in the liver tissues of p21-HBx Tg mice aged 6, 12 and 18 mouths versus WT littermate mice were determined by western blotting, respectively (**P < 0.01, Student’s t test). (B) The integrations of HBx and survivin genes into the genomes of LO2 cells were validated by PCR using genomic DNA as a template. GAPDH was used as a loading control. (C) The effect of HBx and/or survivin on cell proliferation was detected by colony-formation assay (*P < 0.05, **P < 0.01, Student’s t test). (D) Tumor formation in nude mice (n = 8 per group) injected with LO2-X or LO2-X-S cells was assessed in 3 weeks. The expression of AFP was tested in the tumor tissues from mice by western blotting and IHC analysis, respectively.

Figure 2

HBx down-regulates miR-520b through binding to Sp1 with partner survivin. (A) The expression levels of miRNA-520b, miRNA-520e, miRNA-29a and miRNA-181c were examined by qRT-PCR in LO2-X-S/LO2-X cells. (B) The expression of miR-520b in clinical HCC and peritumor samples was detected by qRT-PCR. (C) A model shows Sp1 binding site-directed mutation in the promoter region of miR-520b. (D) The effect of knockdown of Sp1 on miR-520b in LO2-X-S or HepG2.2.15 cells was examined by qRT-PCR analysis (***P < 0.001, Student’s t test). (E) The effect of Sp1 on miR-520b promoter in LO2-X-S cells was tested using Sp1 siRNA (Si-Sp1) or Sp1 mutant by luciferase reporter gene assays (**P < 0.01, Student’s t test). (F-H) The interaction among HBx, survivin and Sp1 in a complex was examined by co-IP. (I) Interaction of the complex, including HBx, survivin and Sp1, with the promoter region of miR-520b was examined by ChIP in LO2-X-S cells.

Figure 3

HBx up-regulates HBXIP in HBx-Tg mice and HCC tissues with partner survivin. (A) The expression of HBXIP was detected by western blotting in LO2 and engineered cell lines (*P < 0.05, Student’s t test). (B) The expression of HBXIP in the liver tissues of p21-HBx-Tg mice aged 6, 12 and 18 mouths versus WT littermate mice were determined by western blotting, respectively (*P < 0.05, Student’s t test). (C) Expression of HBXIP was examined by IHC staining in the clinical tissues of normal liver, hepatitis, liver cirrhosis, HCC and peritumor tissues. (D, E) Correlation between relative expression of HBXIP and that of HBx (or survivin) was examined by qRT-PCR in 22 cases of HCC tissues (***P <0.001, r = 0.797 or r = 0.717; Pearson’s correlation coefficient). Data presented are from three independent experiments.

Figure 4

HBx survivin-dependently up-regulates HBXIP via DNA demethylation of HBXIP. (A) Schematic representation of the HBXIP promoter was shown. The activities of different fragments from HBXIP promoter were tested by luciferase reporter gene assays in HepG2-X, H7402-X and LO2-X-S cells (*P < 0.05, **P < 0.01, Student’s t test). (B, C) The methylation levels of HBXIP CpG sites were examined by bisulfite sequencing and MSP analysis in the cells and clinical tissues (n = 4), respectively. Eight CpG sites were analyzed. Closed and open circle present methylated and unmethylated CpG site, respectively. N represents adjacent nontumorous tissue; T represents HCC tissue. Bands in the ‘M’ and ‘U’ lanes are PCR products obtained with methylation-specific and unmethylation-specific primers, respectively.

Figure 5

MiR-520b/HBXIP modulates proliferation of LO2-X-S cells in vitro. (A-D) The effects of miR-520b, miR-520b/HBXIP or si-HBXIP on proliferation of LO2-X-S cells were examined by MTT, EdU incorporation or colony-formation assays, respectively. Si-NC refers to negative control of HBXIP siRNA. Data are shown as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, Student’s t test).

Figure 6

MiR-520b/HBXIP modulates growth of LO2-X-S cells in vivo. (A-G) The effect of miR-520b or si-HBXIP on the growth of LO2-X-S cells was detected by xenograft assay. The expression levels of miR-520b and HBXIP in the tumor tissues from mice (n = 5, each group) were determined by qRT-PCR or western blot analysis, respectively. Data are shown as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, Student’s t test). (H) A model of HBx accelerating hepatocarcinogenesis with partner survivin.