Figure 5.

Restoring of PTEN expression by depletion of DNMT3A in HCC cells is partly due to methylation of its promoter. (a) Quantitative-PCR analysis of PTEN expression level in DNMT3A-depleted cell line. (b) 5-Aza-2′deoxycytidine restored endogenous PTEN mRNA expression in SMMC-7721 cells. (c) Methylated status evaluation of PTEN promoter region determined by MSP. The presence of visible PCR product in lanes marked “U” indicates the unmethylated gene, whereas the presence of product in lanes marked “M” indicates the methylated gene. (d) Bisulfite sequencing of the selected region of the PTEN promoter in DNMT3A-depleted SMMC-7721 cells. Top, sequences within the fragment include 6 CpG sites (S1, S2, S3, S4, S5, S6). Bottom, the methylation pattern of 6 CpG sites in PTEN promoter region is shown. Ten random clones from DNMT3A-depleted SMMC-7721cell line and control were sequenced to examine the methylated status. Open and closed circles indicate unmethylated and methylated sites, respectively.