Genome-wide profiling of DNA methylation reveals preferred sequences of DNMTs in hepatocellular carcinoma cells.

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in developmental process and diseases including cancer. To elucidate the functional preferred site of DNMTs, we analyzed the feature of distinct methylated sequences and established the defined relationship between DNMTs and preference genomic DNA sequences. Small interfering RNA (siRNA) construct of DNTM1, DNMT3A, and DNMT3B was transfected into the human hepatocellular carcinoma cell line SMMC-7721, respectively. Distinguishing methylated fragments pool was enriched by SHH method in cells which is knocked down DNMT1, DNMT3A, DNMT3B, separately. The defined binding transcription factors (TFs) containing of 5'CpG islands were obtained with bioinformatics software and website. In SMMC-7721 hepatocellular carcinoma (HCC) cell line, DNMT1, DNMT3A, and DNMT3B were specific suppressed by their corresponding siRNA construct, separately. A 46, 42, 67 distinctive methylated fragments from three different DNMTs were evaluated according to genomic DNA database. Those separated fragments were distributed among genomic DNA regions of all chromosome complements, including coding genes, repeat sequences, and genes with unknown function. The majority of coding genes contain CpG islands in their promoter region. Cluster analysis demonstrated all of preference sequences identified by three DNMTs shares their own conserved sequences. In depleting of different DNMTs cells, 80 % of 103 upregulation genes induced by DNMT1 knock-down contain CpG sites; 76 % of 25 upregulation genes induced by DNMT3A knock-down contain CpG sites; 63 % of 126 upregulation genes induced by DNMT3B knock-down contain CpG sites. Our findings suggested that distinctive DNMTs targeted DNA methylation site to their preference sequences, and this targeting might be associated with diverse roles of DNMTs in tumorigenesis. Meanwhile, the analysis of preference sequences provides an alternative way to find out the individual function of DNMTs.

Gene induction and apoptosis in human hepatocellular carci-noma cells SMMC-7721 exposed to 5-aza-2'-deoxycytidine.

BACKGROUND: Aberrant DNA methylation plays a key role in human carcinogenesis. 5-aza-2'-deoxycytidine inhibits DNA methylation and induces the expression of genes putatively silenced by promoter methylation in vitro. There are few studies of the biological and clinical significance of 5-aza-2'-deoxycytidine in human hepatocellular carcinoma. This study explored the mechanism of 5-aza-2'-deoxycytidine targeting transcriptional repressor complexes affecting global gene expression in hepatocellular carcinoma cell line. METHODS: High density oligonucleotide gene expression microarrays were used to examine the effects of 5-aza-2'-deoxycytidine treatments on human hepatocellular carcinoma cell line SMMC-7721. The 5' ends of the genes upregulated or downregulated in this manner were compared with BLAST database to determine whether they might have promoter CpG islands. Flow cytometry was used to detect stages of the cell cycle and apoptosis of SMMC-7721 after being treated with 5-aza-2'-deoxycytidine. RESULTS: Data obtained 3 days after 4 days of treatment with 5-aza-2'-deoxycytidine showed that more genes were induced in tumorigenic cells including genes that function in cell proliferation, differentiation, regulation of transcription, and cytokine signalling. Approximately 30% of induced genes did not have CpG islands within their 5' regions, suggesting that some genes activated by 5-aza-2'-deoxycytidine may not result from the direct inhibition of promoter methylation. This phenomenon may contribute to a number of upregulated genes involving regulation of transcription in the treated cell. Results showed that 100 micromol/L 5-aza-2'-deoxycytidine blocked cell cycle at S/G2-M phase increasing rate of apoptosis. Notably, we found differential expression of molecular action in the methylation although DNA methyltransferases did not show significant difference in the treated cell line. CONCLUSION: 5-aza-2'-deoxycytidine could restore some silenced genes expression independently of DNA methylation inhibition and expression of DNA methyltransferases.