OBJECTIVETo explore the mechanism of hepatitis B virus X protein (HBx)-mediated inhibition of CYP2El expression and its significance in hepatocellular carcinoma (HCC) metastasis.

METHODSA deletion series and mutagenesis series of human CYP2E1 promoter sequence was co-transfected with the HBx expression vector pCMV-2B-FLAG-X into the human HepG2 hepatoma cell line. Reverse transcription-PCR and real-time PCR were used to evaluate the effects of HBx on CYP2E1 promoter activity. The luciferase reporter gene assay was used to identify the HBx-responsive region in the CYP2E1 promoter. Electrophoretic mobility shift assay was used to detect the protein complexes binding to nucleic acids in the CYP2E1 promoter. Martrigel invasion assay was used to examine effects of HBx-inhibited CYP2E1 on invasiveness.

RESULTSAnalysis of the deletion series and mutagenesis series led to identification of two regions of sequence in the CYP2E1 promoter that are important in HBx-mediated modulation of CYP2E1 activity in HepG2 cells (F = 112.24, P = 0.001). Both HNF4alpha and SREBP-1, which directly interact with CYP2E1 promoter sequences, were implicated in the mechanism of HBx-mediated modulation of CYP2E1 promoter activity. In addition, PI3K and JNK pathways were involved in the HBx-mediated modulation (t = 8.56, P = 0.0012 and t = 10.25, P = 0.0009 respectively). HBx-mediated repression of constitutive CYP2E1 led to increased invasiveness.

CONCLUSIONHBx-mediated inhibition of CYP2E1 expression may promote HCC by increasing tumor progression and invasiveness through modulation of the PI3K and JNK signaling pathways.