Figure 1. HBx up-regulates the mEZH2 expression by activating the mEZH2 promoter. A, the expression of mEZH2 is up-regulated by HBx. The HBx expression plasmid (0–1 µg) was transfected into AML12 cells seeded in 24-well plates. When less than 1 µg of the HBx-expressing plasmid was used for transfection, the control vector was added to bring the amount of transfected DNA up to 1 µg. The protein expression level was analyzed by Western blotting 48 h after transfection. B, amplification of mEZH2 promoter sequences. The amplified mEZH2 promoter fragments were separated by agarose electrophoresis. Lane M, Marker III; lane 1, amplified mEZH2 promoter sequence from −2406 to −1517; lane 2, mEZH2 promoter sequence from −1650 to −885; lane 3, mEZH2 promoter sequence from −971 to −215; lane 4, mEZH2 promoter sequence from −333 to +22; lane 5, the whole length of mEZH2 promoter sequence amplified from −2406 to +22. C and D, the activity of mEZH2 promoter is up-regulated by HBx in a dose-dependent manner using luciferase reporter assays. The mEZH2 promoter luciferase reporter vector pGL3-pmEZH2 (−2406/+22) was co-transfected with HBx expression vector pcDNA4.0-EGFP-HBx or control vector pcDNA4.0-EGFP. The pGL3-Basic vector without any promoter sequence was also transfected as a negative control (C). AML12 cells were co-transfected with 0.5 µg of pGL3-pmEZH2 (−2406/+22) and 0 to 1 µg of pcDNA4.0-EGFP-HBx. When less than 1 µg of the pcDNA4.0-EGFP-HBx plasmid was used for transfection, the pcDNA4.0-EGFP vector was added to bring the amount of transfected DNA up to 1 µg (D). The data shown was one result of two independent experiments.