DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation

Abstract

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.

Keywords: CpG island; Methylation; VAV3; cancer; cancer-related genes; de novo methyltransferase; overexpression of DNMT3B.

Figure 1

Gene ontology analysis of downregulated genes by overexpression of DNMT3B in HaCaT cells. A: We used H35K array of 35764 genes, the graph shows the number of genes that change their expression by overexpression of DNMT3B. B: Gene ontology (GO) analysis for downregulated genes by overexpression of DNMT3B.

Figure 2

Prediction of CpG island in downregulated genes by overexpression of DNMT3B in HaCaT cells. A: Classification of downregulated genes according to Z-score value, the graph shows the number of genes for each Z-score range. B: Number of genes with and without CpG island. C: Gene ontology (GO) analysis for 151 genes with CpG island.

Figure 3

Validation of microarray data by RT-qPCR. mRNA quantification of 10 genes in HaCaT cells with overexpression of DNMT3B and control HaCaT cells. The bars represent the mean ± standard deviation from at least three independent experiments. *P < 0.05

Figure 4

Methylation analysis of VAV3 promoter in HaCaT cells. A: Schematic representation of the CpG island and CpG sites in the VAV3 promoter. For methylation analysis the VAV3 promoter was divided into 2 regions: R1 -599 to -307 with 34 CpGs and R2 -299 to +20 with 61 CpGs, the positions are relative to the transcription start site. The primers for MSP and BSP are indicated by black and red arrows, respectively. Each CpG site is represented by a vertical bar. B: The methylation status of the VAV3 promoter (R1 and R2) was determined by MSP in HaCaT cells with overexpression of DNMT3B and control HaCaT cells. U showed unmethylation-specific primer amplification, M showed methylation-specific primer amplification. C: BSP analysis of the VAV3 promoter (R1 and R2) in HaCaT cells with overexpression of DNMT3B and control HaCaT cells. Black circles represent methylated CpG site and white circles represent unmethylated CpG site. The red box shows the two regions more densely methylated by overexpression of DNMT3B.

Figure 5

Expression of DNMT3B, VAV3, SORBS2 and GPR137 in human cancer by RT-qPCR. A: mRNA expression levels of DNMT3B in cervical cancer and normal cervix. The mRNA expression levels of GAPDH were used as internal control. B: mRNA expression levels of DNMT3B, VAV3, SORBS2 and GPR137 in cervical, lung and breast cancer cell lines. The data are presented as the fold change in cancer cell line relative to HaCaT cell line. The bars represent the mean ± standard deviation from at least three independent experiments. *P < 0.05.