Whole DNA methylome profiling in lung cancer cells before and after epithelial-to-mesenchymal transition

Abstract

Background: Metastatic lung cancer is one of the leading causes of cancer death. In recent years, epithelial-to-mesenchymal transition (EMT) has been found to contribute to metastasis, as it enables migratory and invasive properties in cancer cells. Previous genome-wide studies found that DNA methylation was unchanged during EMT induced by TGF-β in AML12 cells. In this study, we aimed to discover EMT-related changes in DNA methylation in cancer cells, which are poorly understood.

Methods: We employed a next-generation sequencing-based method, MSCC (methyl-sensitive cut counting), to investigate DNA methylation during EMT in the A549 lung cancer cell line.

Results: We found that methylation levels were highly correlated to gene expression, histone modifications and small RNA expression. However, no differentially methylated regions (DMRs) were found in A549 cells treated with TGF-β for 4 h, 12 h, 24 h and 96 h. Additionally, CpG islands (CGIs) showed no overall change in methylation levels, and at the single-base level, almost all of the CpGs showed conservation of DNA methylation levels. Furthermore, we found that the expression of DNA methyltransferase 1, 3a, 3b (DNMT1, DNMT3a, DNMT3b) and ten-eleven translocation 1 (TET1) was altered after EMT. The level of several histone methylations was also changed.

Conclusions: DNA methylation-related enzymes and histone methylation might have a role in TGF-β-induced EMT without affecting the whole DNA methylome in cancer cells. Our data provide new insights into the global methylation signature of lung cancer cells and the role of DNA methylation in EMT.

Virtual slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1112892497119603.

Figure 1

Characteristics of the A549 cell line before and after EMT. (a) The morphology of A549 cells treated with TGF-β (5 ng/ml) for different time intervals was visualized by phase-contrast microscopy (Olympus). (b) The expression of E-cadherin, N-cadherin, Vimentin, and Snail in TGF-β-treated A549 cells was analyzed by western blotting. (c) mRNA extracted from A549 cells treated with TGF-β was analyzed by RT-PCR (E-cadherin is shown on the left y-axis, and N-cadherin, Vimentin, and Snail1 are shown on the right y-axis).

Figure 2

Global properties of DNA methylation in the A549 cell line. (a) The distribution of all CCGG sites and those with more than 30 reads. CCGG sites with 30+ reads are similarly distributed with all CCGG sites. (b) The distribution of CCGG methylation levels from the perspective of genes (hollow dots). The black line shows the moving average methylation level. (c) The CCGG sites with 30+ reads were mapped to CGIs and CGI shores. More than half of the CCGG sites are located in CGIs or CGI shores. (d) Overall methylation levels of the CCGG sites located in the respective regions.

Figure 3

DNA methylation levels closely correlate with gene expression, histone modification and short RNA expression. (a) The methylation level of genes with high (red line), medium (orange line) and low (blue line) expression levels. (b) The TSS region and latter half of the gene body show differential DNA methylation levels that correspond to gene expression level. (c) Comparison of DNA methylation levels within and without differentially histone bound regions. The histone modifications analyzed include histone acetylation and methylation. (d) The percentage of CCGG sites located in and near short RNA coding regions. (e) The overall methylation level of CCGG sites that are within, near and without short RNA coding regions.

Figure 4

Comparison of genome-wide DNA methylation of A549 cells before and after EMT. (a) The average methylation level of 200-bp windows (violet red dots) and CGIs (cyan dots) of S4h, S12h, S24h and S96h compared with S0h. A 25% difference boundary line is shown (black line). (b) Example regions to show that the average methylation level of the 200 bp windows is similar in S0h (blue line) and S24h (violet red line). (c) The average methylation level of CGIs that were hypo-, semi- and hypermethylated in primary A549 cells. The data of 100 CGIs are shown as examples.

Figure 5

Expression of methylation-related enzymes and histone methylation levels in A549 cells before and after EMT. Changes in DNMTs, TETs and several types of histone methylation after TGF-β treatment over different time intervals were examined by Western blotting.