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.
DNA methyltranferases Preference sequences 5′CpG site
Small interfering RNA
Subtractive suppression hybridization
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This work was supported by The National Natural Science Foundation of China, No. No.91229107. This work was also supported by National 973 Basic Research Program of China 2013CB911302, No.81171915, No. 81472548 and 30470950.
Conflicts of interest
FH was responsible for designing and execution, collation of study materials, and verification of the data and final manuscript writing; ZZJ designed and performed the siRNA of DNMTs in HCC cell lines and analyzed the microarray data; CH contributed to experiment performing and data collection. CYC designed and performed SSH. QFC, WL, HJJ, WHZ, and SW participated in the completion of the study. All the authors read and approved the final manuscript.
Restoring expression of representative genes by depletion of different DNMTs in HCC SMMC-7721 cells. (A) Analysis of expression level of representative up-regulated gene CDH1 by Quantitative-PCR (left) and Western Blot (right) in DNMT1-depleted cell. (B) Analysis of expression level of representative up-regulated gene PTEN by Quantitative-PCR (left) and Western Blot (right) in DNMT3A-depleted cell. (C) Analysis of expression level of representative up-regulated gene PDCD4 by Quantitative-PCR (left) and Western Blot (right) in DNMT3B-depleted cell. (JPEG 858 kb)
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