Influence of DNA methyltransferase 3b on the expression of cyclin D1 gene and methylation of its promoters in human hepatocellular carcinoma cells

Abstract

To investigate the influence of DNA methyltransferase (DNMT) 3b on the expression of cyclin D1 gene and methylation of its promoters and to investigate the function of DNMT3b. Human hepatocellular carcinoma cells of the line SMMC7721 were cultured and randomly divided into 3 groups: experimental group transfected with siRNA to silence the DNMT3b, control group transfected with control siRNA, and normal group without transfection. The transfection rate of siRNA was detected by fluorescence microscopy. MTT method was used to measure the survival rate of the SMMC-7721 cells. Western blotting and cell proliferation assay were performed to evaluate the expression of cyclin D1 and cell growth. Methylation specific PCR (MSP) was performed to investigate whether the promoter of cyclin D1 was methylated. Fluorescence microscopy showed that the transfection rate of siRNA was over 90%. MTT method showed that 24 h and 36 h after transfection the A value and survival rate of the SMMC7721 cells of the experimental group were both significantly higher than those of the control d normal groups (all P < 0.05). Western blotting showed that the expression levels of DNMT3b and cyclin D1 of the experimental group decreased significantly compared with the control and the normal groups. MSP showed no obvious change of the state of methylation among the 3 groups. DNMT3b may regulate the expression and the function of cyclin D1 gene in the human hepatocellular carcinoma cells, but does not change its methylation state. DNMT3b may play their role as a signal transduction element rather than as a DNA methyltransferase.