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Figure 1 | BMC Cancer

Figure 1

From: Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation

Figure 1

HBx induces DNA methylation in the promoter region of the caveolin-1 gene. (A) There is a CpG island in the promoter region of the caveolin-1 gene. Gene of Interest: The targeted gene that we studied. (B) DNA methylation status in HCC tissues was examined by n-MSP. U: unmethylated PCR products, M: methylated PCR products. 1: healthy liver tissue, 2: M.Sss I-treated healthy liver tissue, 3ā€“9: HCC liver tissues. (C) DNA methylation status in SMMC-7721 cells was examined. 1: healthy liver tissue, 2: M.Sss I-treated healthy liver tissue, 3: mock-treated cells, 4: HBx-transfected cells. (D) Methylated CpG sites in the caveolin-1 promoter region were identified by sequencing. Black boxes represent newly methylated CpG sites after HBx transfection. Red boxes represent de novo methylated CpG sites. U: Pmd-18 T-Cav-U, M: Pmd-18 T-Cav-M. (E) DNA methylation status was examined in SMMC-7721 cells, 1: mock treatment, 2: (Mock-a) mock and 5ā€²-Aza-2ā€²-DC treatment, 3: HBx transfection, 4: (HBx-a) HBx transfection and 5ā€²-Aza-2ā€²-DC treatment. (F) Caveolin-1 mRNA was examined at 48 hours post-infection by RT-PCR, 1: mock treatment, 2: HBx transfection, 3: HBx transfection and 5ā€²-Aza-2ā€²-DC treatment. (G) Caveolin-1 protein expression in SMMC-7721 cells was examined at 48 hours post-infection by western blot. Mock: SMMC-7721 cells were transfected with an empty GFP recombinant adenoviral vector. HBx: HBx transfection, HBx-a: HBx transfection and 5ā€²-Aza-2ā€²-DC treatment. (H) Transcriptional activity of the caveolin-1 promoter was determined by a dual luciferase reporter assay. pGL3-Basic: empty vector, pGL3-Basic-Cavpro: recombinant pGL3-Basic containing the caveolin-1 promoter. (I) Expression of caveolin-1 in HCC and adjacent tissue with HBV infection, as determined by immunohistochemistry. A: adjacent non-tumor tissue, B: HCC. (J) DNA methylation status in adjacent non-tumor tissue and HCC was examined by n-MSP. U: unmethylated PCR products, M: methylated PCR products. 1: healthy liver tissue, 2: M.Sss I-treated healthy liver tissue, 3: adjacent non-tumor tissue, 4: HCC.

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