Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

Figure 1 DNMT1 expression was analyzed in pericancerous liver tissues and HCCs by immunohistochemistry (x 100). A:The expression of DNMT1 was detected in cirrhotic noncancerous liver tissue obtained from HCC patients; B: The expression of DNMT1 was detected in well differentiated HCC; C: The expression of DNMT1 was detected in poorly differentiated HCC. The arrow is to show DNMT1 antibody staining in cell nuclei.

Figure 2 DNMT1 mRNA expression levels analysis in HCC cell lines by quantitative real-time PCR. DNMT1 mRNA expression levels were normalized according to the β-actin mRNA level of the same cell line. DNMT1 was increased more in most hepatocellular carcinoma cell lines, especially in BEL-7402, BBEL-7405 and SMMC-7721, than in the normal cell line HL-7702 and pericarcinoma cell line QSG-7701.

Figure 3 Inhibition of DNMT1 affects cell growth in HCC cell lines. Expression of endogenous DNMT1 protein in 7721-MT1 cells transfected with DNMT1 RNAi construct were decreased more than 80%. A: The SMMC-7721 cells were transfected transiently with DNMT1 siRNA and analyzed for their survival by trypan blue staining assay. The assays were performed in triplicates and the data are the mean ± SE; B: Western blotting analysis of DNMT1 expression in cells transfected transiently with DNMT1 siRNA for 1, 3, 5, 7 d; C: The SMMC-7721 cells were transfected stably with DNMT1 siRNA for 2 mo and the inhibition of DNMT1 and expression of PCNA were evaluated.

Figure 4 Knock-down DNMT1 induces cell apoptosis in HCC cell lines. A: The SMMC-7721 cells were transfected stably with DNMT1 siRNA for 2 mo and apoptosis was detected by FACS; B: Knock-down DNMT1 induced apoptosis gene PDCD4 expression in 7721-MT1 cell line compared with control cell line by quantitative real-time PCR. ^aShow the significant difference between 7721-pMT1 and control.