Fig. 2

FOXC1 induces DNA hypermethylation of CTH promoter and CTH gene silencing through upregulating DNMT3B expression. a BGS detected the methylation status of the CTH promoter in the indicated HCC cells. A typical CpG island is presented at the promoter region of CTH. Each red vertical bar represents a single CpG site. The transcription starts site (TSS) is indicated by a curved arrow. Black spots represent 75–100% methylated CpGs, yellow spots denote 50–75% methylated CpGs, orange spots denote 25–50% methylated CpGs and hollow dots represent 0–25% methylated CpGs. b-c DNA methylation status of the CTH promoter was examined by MSP in the indicated HCC cells and in 10 pairs of HCC tissues (T1-T10) and tumor-adjacent tissues (N1-N10). U, unmethylated DNA; M, methylated DNA. d After the cells were treated with 5-Aza (5 μM, 72 h), western blots were used to detect the protein levels of CTH. e-f Western blotting analysis of DNMT1, DNMT3A, DNMT3B and FOXC1 expression in the indicated cells. g Relative luciferase activity in Huh7 cells after the co-transfection of a construct containing the DNMT3B promoter with an FOXC1-overexpressing construct was examined by luciferase reporter assay. h DNMT3B promoter constructs with serially truncated and mutated sequences which were cloned into pGL3-luciferase reporter plasmids were transfected into Huh7 cells. Then, pCMV-FOXC1 plasmids were co-transfected to determine the relative activity of luciferase. The structure of the diagram is shown (left) and the histogram shows the relative level of luciferase activity in each sample (right). i ChIP assays clarified the direct binding of FOXC1 to the DNMT3B promoter in Huh7-FOXC1 cells (upper panel) and the enriched binding of endogenous FOXC1 to the DNMT3B promoter in primary HCC tissues (lower panel). Hepatocytes were isolated from the liver tissues of HCC patients (n = 6) and healthy controls (n = 3). j Correlation analysis of the expression of CTH and promoter methylation status of CTH in HCC patients in cohort I (left) and cohort II (right). k Kaplan-Meier analysis of the correlation between promoter methylation status of CTH and the recurrence or overall survival in patients with HCC in cohort I (left) and cohort II (right). Data are represented as mean ± S.D. of three experiments. *P < 0.05