DNA methyltransferase 1 knockdown induces silenced CDH1 gene reexpression by demethylation of methylated CpG in hepatocellular carcinoma cell line SMMC-7721

Abstract

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality in the world; however, the molecular mechanisms leading to hepatocyte transformation, especially in epigenetic mechanisms (such as DNA methylation) are still poorly understood. DNA methyltransferase 1 (DNMT1) is the predominant maintenance methyltransferase gene required to maintain DNA methylation patterns in mammalian cells. To explore the role of DNMT1 in the regulation of expression of tumor-related genes in human HCC cells via DNA methylation of the regulatory CpG islands, we stably transfected expression constructs containing small interfering RNA (siRNA) of DNMT1 into the human HCC cell line, SMMC-7721. RNA interference knocked down specific DNMT1 protein expression, resulting in the demethylated promoter of CDH1 and the reexpression of CDH1 in 7721-pMT1. By contrast, promoter methylation and lack of gene expression were maintained when the cell lines had control constructs. Knock down of DNMT1 expression by siRNA induced the promoter of CDH1 demethylation and upregulated CDH1 transcription. High-density oligonucleotide gene expression microarrays were used to examine the effects of DNMT1 knock down on human HCC cells (SMMC-7721); these showed that a number of genes were induced in the DNMT1 knock down cell lines, including some important tumor-related genes such as PDCD4, DCN and PTGES except CDH1. Only approximately 78% of the induced genes have CpG islands within their 5' regions, suggesting that certain genes activated by DNMT1 siRNA might not have resulted from the direct inhibition of promoter methylation. In hepatocellular carcinoma, DNMT1 is necessary to maintain the methylation of CpG islands in certain tumor-related genes.