Fig. 4.

Aberrant CpG methylation of the first intron region of EYA2 down-regulates the expression of mRNA in HCC. (A) mRNA expression of EYA2 in four HCC cell lines by qRT-PCR after treatment with 5-aza-2′-deoxycytidine for 12 h compared with DMSO-treated cells. (B) The methylation level of EYA2 CpG islands in HCC and adjacent tissue (left), and negative correlation between methylation level of EYA2 CpG islands and EYA2 mRNA expression based on MethHC database (right). (C) The location of CpG in EYA2 intron 1 and PCR primers used for the methylation analysis. (D) Profiling of the methylation levels of CpG sites in EYA2 intron 1 presented as a dot chart. (E) Average methylation level of each CpG site in HCC and adjacent tissues (n = 30 pairs). (F) mRNA expression of EYA2 (left) and 10 significantly different CpG methylation sites (right) in 30 paired HCC tissues and adjacent tissues. (G) Correlation analysis between mean methylation of 10 significantly different CpGs and corresponding mRNA expression of EYA2. *P < 0.05, **P < 0.01, ***P < 0.001. (A, B, E, F) mean ± SEM, Student’s t-tests; (B, G) Pearson’s correlation test