HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.
OBJECTIVE
Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2.


METHODS
We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs.


RESULTS
CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB.


CONCLUSIONS
HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC.
Division of Gastroenterology and Hepatology, University of Michigan Health Systems, Ann Arbor, MI, USA.
Yuehua
Y
Huang
Andrew W
AW
Tai
Shuping
S
Tong
Anna S F
AS
Lok
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000209:etiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050687:E2F1 Transcription Factor / Q000502:physiology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000209:etiology; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics* / Q000502:physiology; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic; D015854:Up-Regulation; D054334:Viral Regulatory and Accessory Proteins
D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D044786:S-Phase Kinase-Associated Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein