Promoter Hypomethylation Is Responsible for Upregulated Expression of HAI-1 in Hepatocellular Carcinoma

Figure 2

Epigenetic modification of HAI-1 in HCC cell lines. (a) The cloned HAI-1 promoter DNA fragments. The upper cartoon shows the genomic DNA, including the mRNA start site “0.” The below four lines show various fragments containing 5 truncations of cloned HAI-1 promoter regions (but not the mRNA start site). (b) The luciferase reporter containing fragments after enzyme digestions and ligations was further transfected into Hep3B and SMMC7721 cell lines. (c, d) The relative firefly/Renilla luciferase activities of different HAI-1 promoter fragments in SMMC7721 (c) and Hep3B (d) cell lines. (N.S.: no significant difference; , , and ; .) (e) Schematic structure of the GC content of HAI-1 predicted online (http://www.urogene.org/methprimer2/). The red arrow indicated as the mRNA start site and transcriptional direction. The blue arrow indicated as the site of start codon ATG. (f) BGS region of the HAI-1 CGIs. (g) For each cell line, 20 single clones were selected for sequencing. The average methylation frequency for each CpG site in every HCC cell line. From blue (0% methylated) to red (100% methylated).