Cell Discovery Tang Fuchou's team and Hao Jihui's team work together to reveal the epigenetic regulation mechanism of pancreatic ductal adenocarcinoma

Pancreatic cancer is one of the common digestive tract malignancies, and is known as the king of cancer because of its extremely high mortality rate.
80% to 90% of pancreatic cancers are pancreatic ductal adenocarcinoma (Pancreatic ductal adenocarcinoma, PDAC)
.
Because the pancreas is a digestive organ, and the early diagnosis of pancreatic cancer is very difficult, the degree of malignancy is high, the surgical resection rate is low, and the prognosis is poor, the clinical sampling of pancreatic cancer is more difficult than other cancers, especially in the same patient.

In order to comprehensively reveal the molecular characteristics of pancreatic ductal adenocarcinoma tumor cells, the research group of Tang Fuqiu from Beijing Future Gene Diagnosis Advanced Innovation Center, Peking University Biomedical Frontier Innovation Center, and Hao Jihui's research group from Tianjin Medical University Cancer Hospital cooperated with 13 cases of pancreatic cancer.
The tumor cells of patients with ductal adenocarcinoma underwent high-precision single-cell multi-omics sequencing analysis, and the key features and regulatory relationships of the tumor cell epigenome (DNA methylome and chromatin status group) and transcriptome were systematically analyzed (Fig.
1)
.
The related research results were published online in Cell Discovery in the form of a research paper on February 15, 2022 , entitled Integrated single-cell multiomics analysis reveals novel candidate markers for prognosis in human pancreatic ductal adenocarcinoma .

Figure 1.
Schematic diagram of sampling methods and research ideas for pancreatic ductal adenocarcinoma

The main findings of the study are:

(1) There is a high proportion of normal pancreatic epithelial cells in the primary tumor tissue of pancreatic ductal adenocarcinoma, which provides a precise control for the study of tumor cell characteristics
.

In this study, the researchers optimized the single-cell multi-omics sequencing technology developed in the laboratory.
After magnetic beads separated the nucleus and cytoplasm of a single cell, the cytoplasmic fraction was transcriptome sequenced using STRT-seq and scCOOL- seq sequences parts of the nucleus, enabling simultaneous detection of gene expression, genome copy number variation, DNA methylation, and chromatin accessibility status in a single cell
.
By analyzing transcriptome data, the researchers identified pancreatic epithelial cells and further differentiated tumor epithelial cells from normal epithelial cells based on copy number variation

　　Figure 2.
Characterization of DNA methylation and chromatin status in pancreatic ductal adenocarcinoma tumor cells and normal epithelial cells

　　(2) In pancreatic ductal adenocarcinoma tumor cells, DNA methylation in the promoter regions of hundreds of genes was strongly up-regulated, and the corresponding chromatin changed from an open state to a closed state, and showed a high degree of consistency in all patients
.
These results suggest that the use of DNA methylation inhibitors to reactivate these genes that are commonly silenced by DNA methylation in different patients may be helpful in the treatment of pancreatic ductal adenocarcinoma

　　Although the overall DNA methylation level of pancreatic ductal adenocarcinoma tumor epithelial cells was reduced, the results of differential methylation analysis of gene promoter regions showed that the number of genes whose DNA methylation was up-regulated in gene promoter regions was much higher in tumor cells.
The number of genes was higher than that of down-regulated genes (Fig.
3), indicating that more genes were silenced due to up-regulated promoter methylation in pancreatic ductal adenocarcinoma
.
In different tumor patients, genes that up-regulate promoter methylation are highly consistent, and gene function annotation analysis shows that these genes are mainly related to biological processes such as nervous system function, cell fate and morphogenesis, suggesting that these genes are involved in tumorigenesis likely played an important role in the development

　　Figure 3.
Differentially methylated genes and functional annotations of pancreatic ductal adenocarcinoma tumor cells and normal epithelial cells　　

　　(3) In pancreatic ductal adenocarcinoma tumor cells, the DNA methylation of gene promoters was negatively correlated with the expression of corresponding genes, while the DNA methylation of gene body was enhanced with the expression of corresponding genes.
positive correlation, suggesting that DNA methylation enhances the regulation of gene expression during tumorigenesis
.

　　Through the correlation analysis of transcriptome and DNA methylome in the same single cell, the researchers found that compared with normal epithelial cells, the DNA methylation and RNA expression of the gene body of tumor cells were higher.
A strong positive correlation (Fig.
4), and genes with lower RNA expression levels were also more strongly demethylated in their gene ontology
.
In addition, DNA methylation in gene promoter regions in tumor cells showed a stronger negative correlation with RNA expression

　　Figure 4.
Correlation between gene methylation and corresponding gene expression in pancreatic ductal adenocarcinoma

　　(4) Genome-wide large-scale DNA demethylation in pancreatic ductal adenocarcinoma tumor cells is strongly enriched in heterochromatin regions, indicating that heterochromatin instability is an important molecular feature of pancreatic ductal adenocarcinoma tumorigenesis
.

　　Compared with normal epithelial cells, pancreatic ductal adenocarcinoma tumor cells from different patients experienced different degrees of genome-wide DNA demethylation (except for the P05 patient)
.
The researchers found that demethylation at the genome-wide level of tumor cells was mainly enriched in heterochromatin regions, such as Long interspersed element-1 (LINE-1), which accounted for 17% of the genome (Figure 5)

　　Figure 5.
Genomic demethylation of pancreatic ductal adenocarcinoma tumor cells is enriched in regions of heterochromatin

　　(5) Combined multi-omics analysis revealed key transcription factors that play an important role in the tumorigenesis of pancreatic ductal adenocarcinoma represented by FOSL2 (function enhancement) and ASCL1
(function attenuation) .

　　The researchers conducted a correlation analysis between the degree of chromatin openness and the level of gene expression, and found that the chromatin state of the promoter region was positively correlated with the level of gene expression (Figure 6)
.
64,339 and 47,622 open chromatin regions were identified in tumor cells and normal epithelial cells, respectively, of which about 30% were tumor cell-specific

　　Figure 6.
Enrichment analysis of chromatin open regions and transcription factor binding sequences specific for pancreatic ductal adenocarcinoma tumor cells and normal epithelial cells

　　(6) The prognostic candidate markers of pancreatic ductal adenocarcinoma represented by ZNF667 and ZNF667-AS1
were screened by combined analysis of DNA methylome and transcriptome .

　　Through combined analysis of DNA methylation and gene expression, the researchers found 77 differentially methylated genes of candidate pancreatic ductal adenocarcinoma prognostic and diagnostic molecular markers
.
Among them, ZNF667 and ZNF667-AS1 are marker molecules that have been reported in cancers such as laryngeal squamous cell carcinoma and non-small cell lung cancer
.
Through quantitative analysis of ZNF667 protein in tumor tissues of 98 PDAC patients, the researchers found that patients with high expression of ZNF667 protein had significantly longer survival time
.
In addition, the researchers found that the overexpression of ZNF667 and ZNF667-AS1 genes in various pancreatic cancer cell lines can inhibit tumor cell proliferation, but not affect tumor cell apoptosis, indicating that these two genes are mainly inhibited by inhibiting cell proliferation.
Oncogenesis of pancreatic ductal adenocarcinoma
.

　　Figure 7.
Bioinformatics analysis and functional validation reveal ZNF667 and ZNF667-AS1 as candidate prognostic markers for pancreatic ductal adenocarcinoma

　　In summary, this study used high-precision single-cell multi-omics sequencing analysis to deeply analyze the molecular characteristics of pancreatic ductal adenocarcinoma tumor cells in different omics dimensions and their interrelationships.
A series of potential molecular markers for the prognosis and diagnosis of pancreatic ductal adenocarcinoma, represented by FOSL2, ASCL1, ZNF667 and ZNF667-AS1
, were characterized and provided a reference scheme for the diagnosis and treatment of pancreatic ductal adenocarcinoma .

　　Fan Xiaoying, a researcher at the Guangzhou Laboratory, Lu Ping, a doctoral student at the School of Life Sciences of Peking University, Dr.
Wang Hongwei from Tianjin Medical University Cancer Hospital, Bian Shuhui, a researcher at Nanjing Medical University, and Wu Xinglong, an associate professor at Hebei Agricultural University, are the co-first authors of the paper
.
Professor Tang Fuchou from Beijing Future Genetic Diagnosis Advanced Innovation Center and Peking University Biomedical Frontier Innovation Center and Professor Hao Jihui from Tianjin Medical University Cancer Hospital are the co-corresponding authors of the paper
.
The research project was supported by the National Natural Science Foundation of China, the Beijing Municipal Science and Technology Commission, and the Beijing Future Gene Diagnosis Advanced Innovation Center
.

　　Paper link: https://