Background and overview
Whole-genome bisulfite sequencing (WGBS) is considered as the "gold standard" for methylation sequencing. The principle is that it is treated with Bisulfite, and the C base which is not methylated in the genome is converted into U, and after PCR amplification, it becomes T, which is distinguished from the C base which has methylation modification, and is combined with high throughput sequencing technology, which can be used to determine whether a CpG/CHG/CHH site is methylated, namely to be used to map a single-base resolution genome-wide DNA methylation map.
Mapping of epigenetic features of tumor microenvironment using whole genome bisulfite sequencing:
The development and progression of solid tumors involves dynamic interactions between tumor epithelial cells and their microenvironment. Currently, most epigenetic studies on tumors focus on epithelial tumor cells. There are few studies on the molecular characteristics of the tumor microenvironment that maintains the tumor phenotype. Tumor-associated fibroblasts (CAF) are a major group of tumor microenvironments. CAF can promote the migration and proliferation of prostate cancer cells. The epigenetic signature of CAF is unclear.
In this paper, WGBS technology was used to detect methylation of tumor-associated fibroblast CAF and non-malignant prostate fibroblast NPF, and the differential methylation region DMR between the two was screened and DMR annotation was performed. The results show that DMR enrichment in CAF occurs in the gene regulatory region, and the combined analysis with RNAseq demonstrates that these DMRs regulate the transcription of genes. In addition, the DMR gene annotation results in CAF show that DMR is enriched for tumor-associated signaling pathways. These DMRs screened in CAF can be used to diagnose prostate cancer.
- Non-malignant prostate fibroblast NPF was isolated from 4 cases of prostate cancer isolated from CAF and 4 adjacent tissues. Whole genome methylation sequencing (WGBS) was performed, and the average sequencing depth of each sample was > 7x. The results show that NPF and CAF have similar methylation profiles.
- A comparison of NPF and CAF showed that there were 7,534 DMRs (differential methylation regions) between the two groups, with CAF having more hypomethylated regions (5038).
- It is found that the DMR identified in CAF is enriched in the regulatory region of the gene. The main features include: most of it is outside the CpG island region; the hypomethylated DMR is more located in the promoter region and closer to the transcription initiationSite (TSS); DMR is enriched in the enhancement sub-region.
- Gene annotation of DMR, 30% of DMR is enriched for tumor-associated signaling pathways.
- To further investigate the role of DMR in the regulatory region, RNAseqwas used to detect gene expression levels. The results showed that 445 MDRs were significantly associated with 220 DEGs (differentially expressed genes), of which 162 DMR were associated with up-regulation of DEG. And this correlation can be verified by BSP and RT-PCR.
- Compare methylation data in prostate cancer epithelial cells and prostate cancer tissues in the TCGA database, of which 50 hypermethylated DMRs and 15 hypomethylated DMRs can be considered tumor-specific DMRs for prostate cancer Diagnosis (AUC > 0.8).
- Pidsley R, Lawrence MG, Zotenko E, et al. Enduring epigenetic landmarks define the cancer microvironment. Genome Res 2018; 28(5): 625-638. (IF: 10.101)